We resequenced 876 short fragments in a sample of 96 individuals of Arabidopsis thaliana that included stock center accessions as well as a hierarchical sample from natural populations. Although A. thaliana is a selfing weed, the pattern of polymorphism in general agrees with what is expected for a widely distributed, sexually reproducing species. Linkage disequilibrium decays rapidly, within 50 kb. Variation is shared worldwide, although population structure and isolation by distance are evident. The data fail to fit standard neutral models in several ways. There is a genome-wide excess of rare alleles, at least partially due to selection. There is too much variation between genomic regions in the level of polymorphism. The local level of polymorphism is negatively correlated with gene density and positively correlated with segmental duplications. Because the data do not fit theoretical null distributions, attempts to infer natural selection from polymorphism data will require genome-wide surveys of polymorphism in order to identify anomalous regions. Despite this, our data support the utility of A. thaliana as a model for evolutionary functional genomics.

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Hereditary xerocytosis (HX, MIM 194380)is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration. Copy number analyses, linkage studies, and exome sequencing were used to identify novel mutations affecting PIEZO1, encoded by the FAM38A gene, in 2 multigenerational HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation with the disease phenotype in all affected persons in both kindreds. All patients were heterozygous for FAM38A mutations, except for 3 patients predicted to be homozygous by clinical and physiologic studies who were also homozygous at the DNA level. The FAM38A mutations were both in residues highly conserved across species and within members of the Piezo family of proteins. PIEZO proteins are the recently identified pore-forming subunits of channels that mediate mechanotransduction in mammalian cells. FAM38A transcripts were identified in human erythroid cell mRNA, and discovery proteomics identified PIEZO1 peptides in human erythrocyte membranes. These findings, the first report of mutation in a mammalian mechanosensory transduction channel-associated with genetic disease, suggest that PIEZO proteins play an important role in maintaining erythrocyte volume homeostasis. IntroductionHereditary xerocytosis (also known as HX or dehydrated stomatocytosis, DHSt; OMIM 194380) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration. 1 HX erythrocytes exhibit decreased total cation and potassium content that are not accompanied by a proportional net gain of sodium and water. HX patients typically exhibit mild to moderate, compensated hemolytic anemia. Erythrocyte mean corpuscular hemoglobin concentration is increased and erythrocyte osmotic fragility is decreased, both reflecting cellular dehydration.A locus for HX on chromosome 16 (16q23-q24) was first identified in a large, 3-generation Irish family. 2 This locus was refined to D16S511-16qter via study of 10 kindreds with variants of HX, pseudohyperkalemia, or nonimmune hydrops fetalis. 3,4 Recent studies of one of the original HX families from Rochester, NY, 5 and of a large HX family from Manitoba, Canada 6 confirmed the linkage of the disease phenotype to chromosome 16q, and refined the candidate region to 16q24.2-16qter, a 2.4 million-bp interval containing 51 known or predicted genes. 6 To identify the HXassociated genetic locus, we performed high-resolution single nucleotide polymorphism (SNP) typing and whole-exome sequencing on selected persons from both the New York and Canadian HX kindreds.In the refined candidate region, no regions of copy number variation were detected at 16q24.2-16qter. A large region of homozygosity was detected in this region in DNA from a presumed homozygote from the New York kindred. Exome sequencing identified novel mutations affecting PIEZO1 (encoded by the FAM38A gene) in both HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation with the disease phenotype in all...

In wild-type Saccharomyces cerevisiae, replication forks slowed during their passage through telomeric C1–3A/TG1–3 tracts. This slowing was greatly exacerbated in the absence of RRM3, shown here to encode a 5′ to 3′ DNA helicase. Rrm3p-dependent fork progression was seen at a modified Chromosome VII-L telomere, at the natural X-bearing Chromosome III-L telomere, and at Y‘-bearing telomeres. Loss of Rrm3p also resulted in replication fork pausing at specific sites in subtelomeric DNA, such as at inactive replication origins, and at internal tracts of C1–3A/TG1–3 DNA. The ATPase/helicase activity of Rrm3p was required for its role in telomeric and subtelomeric DNA replication. Because Rrm3p was telomere-associated in vivo, it likely has a direct role in telomere replication.

Mutations in the yeast Saccharomyces cerevisiae PIF1 gene, which encodes a 5'-to-3' DNA helicase, cause telomere lengthening and a large increase in the formation rate of new telomeres. Here, we show that Pif1p acts by inhibiting telomerase rather than telomere-telomere recombination, and this inhibition requires the helicase activity of Pif1p. Overexpression of enzymatically active Pif1p causes telomere shortening. Thus, Pif1p is a catalytic inhibitor of telomerase-mediated telomere lengthening. Because Pif1p is associated with telomeric DNA in vivo, its effects on telomeres are likely direct. Pif1p-like helicases are found in diverse organisms, including humans. We propose that Pif1p-mediated inhibition of telomerase promotes genetic stability by suppressing telomerase-mediated healing of double-strand breaks.

SUMMARY Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4–5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state, and suggest that cell cycle acceleration toward a critical threshold is an important bottleneck for reprogramming.

Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-␤1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation.

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.A llocation of blastomeres to outside and inside positions during preimplantation mammalian development initiates specification of the first two cell lineages, the trophectoderm (TE) and the inner cell mass (ICM) (1, 2). Successful progression of TE and ICM fate specification and proper development of the preimplantation embryo depends on differential transcriptional programs that are instigated and maintained within the outer and inner cells. Gene-KO studies in mice showed TEAD4 as the master orchestrator of the TE-specific transcriptional program (3-5). TEAD4-null embryos do not mature to the blastocyst stage and TEAD4-null blastomeres lack expression of TE-specific master regulators like CDX2, GATA3, and EOMES (3, 4). However, they maintain expression of ICM-specific factors like OCT4 and NANOG.Interestingly, TEAD4 expression is maintained both in cells of TE and ICM lineages, as well as in the TE-derived trophoblast stem cells (TSCs) and ICM-derived ES cells (ESCs) (5, 6). Thus, questions are raised as to how TEAD4 selectively orchestrates the TE/TSC-specific transcriptional program but not the ICM/ ESC-specific transcriptional program. The current model predicts that the presence vs. the absence of a TEAD4 cofactor, yesassociated protein (YAP), modulates TEAD4 function at its target genes in outer vs. inner blastomeres (6), leading to the segregation of the TE and ICM lineages. However, YAP-null mouse embryos do not show preimplantation developmental defects (7), indicating that, unlike TEAD4, YAP function is dispensable during TE and ICM fate determination. It is proposed that another YAP-related cofactor, WWTR1 (i.e., TAZ), could compensate for the absence of YAP during early development (6). However, the mode of TAZ function during TE and ICM specification is unknown. Furthermore, direct targets of TEAD4 have not been identified in the TE or in trophoblast cells. Thus, definitive experiments have not been performed to conclude that loss of cofactor function/recruitmen...