Biology subjects

Markowitz, T. E.

Publications and source records attributed to Markowitz, T. E..

3 recordsLinked to original sources

bioRxiv2018

SNP-ChIP: A versatile and tag-free method to quantify changes in protein binding across the genome

Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) is the method of choice for mapping genome-wide binding of chromatin-associated factors. However, broadly applicable methods for between-sample comparisons are lacking. Here, we introduce SNP-ChIP, a method that leverages small-scale intra-species polymorphisms, mainly SNPs, for quantitative spike-in normalization of ChIP-seq results. Sourcing spike-in material from the same species ensures antibody cross-reactivity and physiological coherence, thereby eliminating two central limitations of traditional spike-in approaches. We show that SNP-ChIP is robust to changes in sequencing depth and spike-in proportions, and reliably identifies changes in overall protein levels, irrespective of changes in binding distribution. Application of SNP-ChIP to test cases from budding yeast meiosis allowed discovery of novel regulators of the chromosomal protein Red1 and quantitative analysis of the DNA-damage associated histone modification{gamma} -H2AX. SNP-ChIP is fully compatible with the intra-species diversity of humans and most model organisms and thus offers a general method for normalizing ChIP-seq results.

genomics↗

bioRxiv2017

Persistent DNA-break potential near telomeres increases initiation of meiotic recombination on small chromosomes

Faithful meiotic chromosome inheritance and fertility relies on the stimulation of meiotic crossover recombination by potentially genotoxic DNA double-strand breaks (DSBs). To avoid excessive damage, feedback mechanisms down-regulate DSBs on chromosomes that have successfully initiated crossover repair. In Saccharomyces cerevisiae, this regulation requires the removal of the conserved DSB-promoting protein Hop1/HORMAD during chromosome synapsis. Here, we identify privileged end-adjacent regions (EARs) spanning roughly 100 Kb near all telomeres that escape DSB downregulation. These regions retain Hop1 and continue to break in pachynema despite normal synaptonemal complex deposition. Differential retention of Hop1 requires the disassemblase Pch2/TRIP13, which preferentially removes Hop1 from telomere-distant sequences, and is modulated by the histone deacetylase Sir2 and the nucleoporin Nup2. Importantly, the uniform size of EARs among chromosomes contributes to disproportionately high DSB and repair signals on short chromosomes in pachynema, suggesting that EARs partially underlie the curiously high recombination rate of short chromosomes.

genomics↗

bioRxiv2017

Acute condensin depletion causes genome decompaction without altering the level of global gene expression in Saccharomyces cerevisiae

Condensins are broadly conserved chromosome organizers that function in chromatin compaction and transcriptional regulation, but to what extent these two functions are linked has remained unclear. Here, we analyzed the effect of condensin inactivation on genome compaction and global gene expression in the yeast Saccharomyces cerevisiae. Spike-in-controlled 3C-seq analysis revealed that acute condensin inactivation leads to a global decrease in close-range chromosomal interactions as well as more specific losses of homotypic tRNA gene clustering. In addition, a condensin-rich topologically associated domain between the ribosomal DNA and the centromere on chromosome XII is lost upon condensin inactivation. Unexpectedly, these large-scale changes in chromosome architecture are not associated with global changes in transcript levels as determined by spike-in-controlled mRNA-seq analysis. Our data suggest that the global transcriptional program of S. cerevisiae is resistant to condensin inactivation and the associated profound changes in genome organization.\n\nSignificance StatementGene expression occurs in the context of higher-order chromatin organization, which helps compact the genome within the spatial constraints of the nucleus. To what extent higher-order chromatin compaction affects gene expression remains unknown. Here, we show that gene expression and genome compaction can be uncoupled in the single-celled model eukaryote Saccharomyces cerevisiae. Inactivation of the conserved condensin complex, which also organizes the human genome, leads to broad genome decompaction in this organism. Unexpectedly, this reorganization has no immediate effect on the transcriptome. These findings indicate that the global gene expression program is robust to large-scale changes in genome architecture in yeast, shedding important new light on the evolution and function of genome organization in gene regulation.

genomics↗