Graphical Abstract Highlights d Early replicating control elements (ERCEs) regulate replication timing d ERCEs regulate A/B compartmentalization and TAD architecture d ERCEs form CTCF-independent loops and have features of enhancer/promoters d ERCEs enable genetic dissection of large-scale chromosome structure and function SUMMARYThe temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide earlyto-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these ''early replication control elements'' (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.

The Ume6 transcription factor in yeast is known to both repress and activate expression of diverse genes during growth and meiotic development. To obtain a more complete profile of the functions regulated by this protein, microarray analysis was used to examine transcription in wild-type and ume6⌬ diploids during vegetative growth in glucose and acetate. Two different genetic backgrounds (W303 and SK1) were examined to identify a core set of strain-independent Ume6-regulated genes. Among genes whose expression is controlled by Ume6 in both backgrounds, 82 contain homologies to the Ume6-binding site (URS1) and are expected to be directly regulated by Ume6. The vast majority of those whose functions are known participate in carbon͞nitrogen metabolism and͞or meiosis. Approximately half of the Ume6 direct targets are induced during meiosis, with most falling into the early meiotic expression class (cluster 4), and a smaller subset in the middle and later classes (clusters 5-7). Based on these data, we propose that Ume6 serves a unique role in diploid cells, coupling metabolic responses to nutritional cues with the initiation and progression of meiosis. Finally, expression patterns in the two genetic backgrounds suggest that SK1 is better adapted to respiration and W303 to fermentation, which may in part account for the more efficient and synchronous sporulation of SK1.

The DNA-binding protein Ume6 is required for both repression and activation of meiosis-specific genes, through interaction with the Sin3 corepressor and Rpd3 histone deacetylase and the meiotic activator Ime1. Here we show that fusion of a heterologous activation domain to Ume6 is unable to convert it into a constitutive activator of early meiotic gene transcription, indicating that an additional function is needed to overcome repression at these promoters. Mutations in UME6 allowing the fusion to activate lie in a predicted amphipathic alpha helix and specifically disrupt interaction with Sin3 but not with Teal, an activator of Ty transcription also found to interact with Ume6 in a two-hybrid screen. The mutations cause a loss of repression by Ume6 and precisely identify the Ume6 Sin3-binding domain, which we show interacts with the paired amphipathic helix 2 region of Sin3. Analysis of these mutants indicates that conversion of Ume6 to an activator involves two genetically distinct steps that act to relieve Sin3-mediated repression and provide an activation domain to Ume6. The mutants further demonstrate that premature expression and lack of subsequent rerepression of Ume6-Sin3-regulated genes are not deleterious to meiotic progression and suggest that the essential role of Sin3 in meiosis is independent of Ume6. The model for Ume6 function arising from these studies indicates that Ume6 is similar in many respects to metazoan regulators that utilize Sin3, such as the Myc-Mad-Max system and nuclear hormone receptors, and provides new insights into the control of transcriptional repression and activation by the Ume6-URS1 regulatory complex in yeast.Saccharomyces cerevisiae adapts to changing environmental conditions by initiating new programs of gene expression that alter the normal progression of mitosis. Depending on cell type and nutritional cues, budding cells switch to a form of invasive pseudohyphal growth, mate to form diploids, stably arrest in stationary phase, or undergo meiosis to form spores (see reference 48, 50, 61, and 91 for reviews). Sporulation typically occurs in MATa/MAT␣ diploids starved for nitrogen and glucose in the absence of a fermentable carbon source and involves the coordinated expression of over 500 genes (13,63). Analysis of this process in yeast is providing important new insights into common strategies and components that regulate not only meiosis and gametogenesis but also cell proliferation and differentiation in multicellular organisms.Meiosis-specific genes in yeast have been divided into several classes depending on their time of expression (13,48,84). UME6 is one of nine UME genes (UME1 to UME9) whose loss causes unscheduled meiotic expression of early meiotic genes during vegetative growth (76, 77; B. Washburn, unpublished data). Although it was originally identified as a transcriptional repressor of early genes, UME6 has also been shown to function in their meiosis-specific activation (9,65,74). UME6 encodes a C 6 zinc cluster protein that binds to the URS1 ciselements fo...