In eukaryotic cells, cohesin holds sister chromatids together until they separate into daughter cells during mitosis. We have used chromatin immunoprecipitation coupled with microarray analysis (ChIP chip) to produce a genome-wide description of cohesin binding to meiotic and mitotic chromosomes of Saccharomyces cerevisiae. A computer program, PeakFinder, enables flexible, automated identification and annotation of cohesin binding peaks in ChIP chip data. Cohesin sites are highly conserved in meiosis and mitosis, suggesting that chromosomes share a common underlying structure during different developmental programs. These sites occur with a semiperiodic spacing of 11 kb that correlates with AT content. The number of sites correlates with chromosome size; however, binding to neighboring sites does not appear to be cooperative. We observed a very strong correlation between cohesin sites and regions between convergent transcription units. The apparent incompatibility between transcription and cohesin binding exists in both meiosis and mitosis. Further experiments reveal that transcript elongation into a cohesin-binding site removes cohesin. A negative correlation between cohesin sites and meiotic recombination sites suggests meiotic exchange is sensitive to the chromosome structure provided by cohesin. The genome-wide view of mitotic and meiotic cohesin binding provides an important framework for the exploration of cohesins and cohesion in other genomes.

The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms. A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals. Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion. Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding. Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion. We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.

Chromosome segregation, transcriptional regulation, and repair of DNA double-strand breaks require the cohesin protein complex. Cohesin holds the replicated chromosomes (sister chromatids) together to mediate sister chromatid cohesion. The mechanism of how cohesion is established is unknown. We found that in budding yeast, the head domain of the Smc3p subunit of cohesin is acetylated by the Eco1p acetyltransferase at two evolutionarily conserved residues, promoting the chromatin-bound cohesin to tether sister chromatids. Smc3p acetylation is induced in S phase after the chromatin loading of cohesin and is suppressed in G(1) and G(2)/M. Smc3 head acetylation and its cell cycle regulation provide important insights into the biology and mechanism of cohesion establishment.

Faithful chromosome segregation and repair of DNA double-strand breaks (DSBs) require cohesin, the protein complex that mediates sister-chromatid cohesion. Cohesion between sister chromatids is thought to be generated only during ongoing DNA replication by an obligate coupling between cohesion establishment factors such as Eco1 (Ctf7) and the replisome. Using budding yeast, we challenge this model by showing that cohesion is generated by an Eco1-dependent but replication-independent mechanism in response to DSBs in G(2)/M. Furthermore, our studies reveal that Eco1 has two functions: a cohesive activity and a conserved acetyltransferase activity, which triggers the generation of cohesion in response to the DSB and the DNA damage checkpoint. Finally, the DSB-induced cohesion is not limited to broken chromosomes but occurs also on unbroken chromosomes, suggesting that the DNA damage checkpoint through Eco1 provides genome-wide protection of chromosome integrity.

In eukaryotes, the process of sister chromatid cohesion holds the two sister chromatids (the replicated chromosomes) together from DNA replication to the onset of chromosome segregation. Cohesion is mediated by cohesin, a four-subunit SMC (structural maintenance of chromosome) complex. Cohesin and cohesion are required for proper chromosome segregation, DNA repair, and gene expression. To carry out these functions, cohesion is regulated by elaborate mechanisms involving a growing list of cohesin auxiliary factors. These factors control the timing and position of cohesin binding to chromatin, activate chromatin-bound cohesin to become cohesive, and orchestrate the orderly dissolution of cohesion. The 45-nm ringlike architecture of soluble cohesin is compatible with dramatically different mechanisms for both chromatin binding and cohesion generation. Solving the mechanism of cohesion and its complex regulation presents significant challenges but offers the potential to provide important insights into higher-order chromosome organization and chromosome biology.

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (ncRNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome.

Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5’ leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression.

Cell differentiation programs require dynamic regulation of gene expression. During meiotic prophase in Saccharomyces cerevisiae, expression of the kinetochore complex subunit Ndc80 is downregulated by a 5’ extended long undecoded NDC80 transcript isoform. Here we demonstrate a transcriptional interference mechanism that is responsible for inhibiting expression of the coding NDC80 mRNA isoform. Transcription from a distal NDC80 promoter directs Set1-dependent histone H3K4 dimethylation and Set2-dependent H3K36 trimethylation to establish a repressive chromatin state in the downstream canonical NDC80 promoter. As a consequence, NDC80 expression is repressed during meiotic prophase. The transcriptional mechanism described here is rapidly reversible, adaptable to fine-tune gene expression, and relies on Set2 and the Set3 histone deacetylase complex. Thus, expression of a 5’ extended mRNA isoform causes transcriptional interference at the downstream promoter. We demonstrate that this is an effective mechanism to promote dynamic changes in gene expression during cell differentiation.