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The establishment of transcriptional silencing in yeast requires cell-cycle progression, but the nature of this requirement is unknown. Sir2 is a protein deacetylase that is required for gene silencing in yeast. We have used temperature-sensitive alleles of the SIR2 gene to assess Sir2's contribution to silencing as a function of the cell cycle. When examined in vivo, these conditional alleles fall into two classes: one class exhibits a loss of silencing when raised to the nonpermissive temperature regardless of cell-cycle position, while the second class exhibits a mitosis-specific silencing defect. Alleles of the first class have a primary defect in protein deacetylase activity, while the alleles of the second class are specifically defective in Sir2-Sir4 interactions at nonpermissive temperatures. Using a SIR2 temperature-sensitive allele, we show that silencing can be established at the HML locus during progression through the [G.sub.2]/M-[G.sub.1] interval. These results suggest that yeast heterochromatin undergoes structural transitions as a function of the cell cycle and support the existence of a critical assembly step for silent chromatin in mitosis.

Heterochromatin regulation of gene expression exhibits epigenetic inheritance, in which some feature of the structure is retained and can reseed formation in new cells. To understand the cell-cycle events that influence heterochromatin assembly and maintenance in budding yeast, we have conducted two types of experiments. First we have examined the kinetics of heterochromatin spreading at telomeres. We have constructed a strain in which the efficient silencing of a telomere-linked URA43 gene depends on the inducible expression of the Sir3 silencing factor. Prior studies determined that S-phase passage was required for the establishment of silencing at the HM loci in yeast. We find that establishment of silencing in our strain occurs at a point coincident with mitosis and does not require S-phase passage. In addition, we find that passage through mitosis is sufficient to establish silencing at the HML locus in a strain bearing a conditional allele of SIR3. Finally, we have also assessed the stability of yeast heterochromatin in the absence of the cis-acting elements required for its establishment. We show that silencing is stable through S phase in the absence of silencers and therefore possesses the ability to self-propagate through DNA replication. However, silencing is lost in the absence of silencers during progression through M phase. These experiments point to crucial events in mitosis influencing the assembly and persistence of heterochromatin.

The usefulness of human induced pluripotent stem cells (hiPSCs) in research and therapeutic applications highly relies on their genomic integrity and stability. Many laboratories including ours have addressed this concern by comparing genomic (at both karyotypic and subkaryotypic levels) and epigenomic abnormalities of hiPSC lines (derived via either DNA- or non-DNA-based methods), as well as human embryonic stem cell lines during long-term culture. A variety of methods have been used for this purpose, such as karyotyping and fluorescent in situ hybridization to detect karyotypic abnormalities, array-based comparative genomic hybridization to detect copy number variations (CNVs), single-nucleotide polymorphism-based microarrays to detect both CNVs and loss of heterozygosity, analysis of integration sites in the genome, and whole genome sequencing for protein-coding exome and DNA methylome profiling. Here, we summarize the progresses in this dynamically evolving field and also discuss how the findings apply to the study and application of hiPSCs. S TEM C ELLS 2012;30:22-27 [ABSTRACT FROM AUTHOR]

Background: Directed differentiation of human induced pluripotent stem cells (hiPSC) into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation. Methodology/Principal Findings: We analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE) cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC) in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons. Conclusions/Significance: Our results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders. [ABSTRACT FROM AUTHOR]

Single-cell adaptive immune receptor repertoire sequencing (scAIRR-seq) offers the possibility to access the nucleotide sequences of paired receptor chains from T-cell receptors (TCR) or B-cell receptors (BCR ). Here we describe two protocols and the downstream bioinformatic approaches that facilitate the integrated analysis of paired T-cell receptor (TR ) alpha/beta (TRA /TRB ) AIRR-seq, RNA sequencing (RNAseq), immunophenotyping, and antigen-binding information. To illustrate the methodologies with a use case, we describe how to identify, characterize, and track SARS-CoV-2-specific T cells over multiple time points following infection with the virus. The first method allows the analysis of pools of memory CD8 + cells, identifying expansions and contractions of clones of interest. The second method allows the study of rare or antigen-specific cells and allows studying their changes over time., (© 2022. The Author(s).)