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Embryonic stem (ES) cells retain the ability to undergo lineage-specific differentiation that can eventually give rise to different cell types that constitute an organism. Although stem cell specific biological networks of transcription factors and epigenetic modifiers are well established, how the ES cell specific transcriptional and alternative splicing (AS) machinery regulate their expression has not been sufficiently explored. In this study, we show that the lncRNA associated protein TOBF1 regulates the co-transcriptional alternative splicing of transcripts necessary for maintaining stem cell identity in mouse ES cells. Overlaying information derived from TOBF1 chromatin occupancy, the distribution of its pluripotency-associated OCT-SOX binding motifs, and transcripts undergoing differential expression and alternative splicing upon its disruption unmasked local nuclear territories where these distinct events converge, ultimately leading to the maintenance of mouse ES cell identity.

Cis-regulatory enhancers are essential for differential expression of developmental and housekeeping genes. However, the specificity of native mammalian enhancers and how it shapes cell-type-specific gene expression landscapes remain largely unknown. We show that endogenous enhancers are broadly compatible with the promoters of developmental and housekeeping genes. Broad enhancer compatibility affords ‘retrofitting’ new regulatory capabilities to housekeeping genes that evolved before the advent of enhancers. This enables cell-type-specific tuning of ubiquitously expressed genes. Segregation between enhancer-dependent and –independent type regulation is blurred. Within the same cell type, a single promoter can be activated by enhancers and non-enhancer promoter-regulatory elements (PREs). It is the tunable and integrated strengths of enhancers and PREs that quantitatively shape gene expression landscapes, within and across cell types. Our findings have broad implications for understanding cell-type-specific quantitative gene expression variation, as well as the emergence and rewiring of gene regulatory networks in disease and organismal evolution.

Chromatin features are widely used for genome-scale mapping of enhancers. However, discriminating active enhancers from other cis-regulatory elements, predicting enhancer strength, and identifying their target genes remains challenging. Here we establish histone H2B N-terminus multisite lysine acetylation (H2BNTac) as a genuine signature of active enhancers. H2BNTac prominently marks candidate active enhancers and their target promoters and discriminates them from ubiquitously active promoters. Two mechanisms afford the distinct H2BNTac specificity. (1) Unlike H3K27ac, H2BNTac is specifically catalyzed by CBP/p300. (2) H2A-H2B, but not H3-H4, are rapidly exchanged through transcription-induced nucleosome remodeling. H2BNTac-positive candidate enhancers show a high validation rate in orthogonal enhancer activity assays, and a vast majority of endogenously active enhancers are marked by H2BNTac and H3K27ac. Notably, H2BNTac intensity predicts enhancer strength and outperforms the current state-of-the-art models in predicting enhancer target genes. These findings have broad implications for generating fine-grained enhancer maps and modeling enhancer-dependent gene regulation.

While current combined antiretroviral therapy (cART) allows control of HIV replication in patients and effectively suppresses plasma viral loads, it is unable to target latent reservoirs, which are responsible for virus rebound after discontinuation of therapy. Several histone deacetylase inhibitors (HDACIs) have been shown to target reservoirs and to reactivate latent HIV. While this effect is highly desired, it carries the risk that HIV-1 may be reactivated in tissue compartments were cART concentrations are insufficient and thus leading tode novoinfections in this sites. To address this concern, we evaluated the effect of different HDACIs for their ability to reverse HIV latency and to modulatede novoinfections. Two of the inhibitors, sodium butyrate and bufexamac, significantly inhibitedde novoHIV-1 infection in activated CD4+T-cells. Transcriptome and proteome analysis indicated global changes of protein abundancies, exhibited reduced proliferation of CD4+T-cells, and revealed butyrate-based proteasomal degradation of EP300, an important factor for HIV-1 replication. Our results disclose new potential treatment strategies and minimizes the concern of potential reservoir reseeding by HDACIs.

The MCM2-7 (minichromosome maintenance) protein complex is a DNA unwinding motor required for the eukaryotic genome duplication1. Although a huge excess of MCM2-7 is loaded onto chromatin in G1 phase to form pre-replication complexes (pre-RCs), only 5-10 percent are converted into a productive CDC45-MCM-GINS (CMG) helicase in S phase – a perplexing phenomenon often referred to as the ‘MCM paradox’2. Remaining pre-RCs stay dormant but can be activated under replication stress (RS)3. Remarkably, even a mild reduction in MCM pool results in genome instability4, 5, underscoring the critical requirement for high-level MCM maintenance to safeguard genome integrity across generations of dividing cells. How this is achieved remains unknown. Here, we show that for daughter cells to sustain error-free DNA replication, their mothers build up a stable nuclear pool of MCMs both by recycling of chromatin-bound MCMs (referred to as parental pool) and synthesizing new MCMs (referred to as nascent pool). We find that MCMBP, a distant MCM paralog6, ensures the influx of nascent MCMs to the declining recycled pool, and thereby sustains critical levels of MCMs. MCMBP promotes nuclear translocation of nascent MCM3-7 (but not MCM2), which averts accelerated MCM proteolysis in the cytoplasm, and thereby fosters assembly of licensing-competent nascent MCM2-7 units. Consequently, lack of MCMBP leads to reduction of nascent MCM3-7 subunits in mother cells, which translates to poor MCM inheritance and grossly reduced pre-RCs formation in daughter cells. Unexpectedly, whereas the pre-RC paucity caused by MCMBP deficiency does not alter the overall bulk DNA synthesis, it escalates the speed and asymmetry of individual replisomes. This in turn increases endogenous replication stress and renders cells hypersensitive to replication perturbations. Thus, we propose that surplus of MCMs is required to safeguard replicating genomes by modulating physiological dynamics of fork progression through chromatin marked by licensed but inactive MCM2-7 complexes.

The multisubunit ubiquitin ligase APC/C (anaphase promoting complex/cyclosome) is essential for mitosis by promoting timely degradation of cyclin B1. Proper timing of APC/C activation is regulated by the spindle assembly checkpoint (SAC), which is initiated by the kinase MPS1 and implemented by MAD2-dependent inhibition of the APC/C. Here we analysed the contribution of the higher eukaryote-specific APC/C subunits APC7 and APC16 to APC/C composition, function and regulation. APC16 is required for APC7 assembly into the APC/C, while APC16 assembles independently of APC7. ΔAPC7 and ΔAPC16 cells display no major defects in mitotic progression, cyclin B1 degradation or SAC response. Strikingly, however, deletion of either APC7 or APC16 is sufficient to provide synthetic viability to MAD2 deletion. ΔAPC7ΔMAD2 cells display an accelerated mitosis and require SAC-independent MPS1 function for maintaining their genome stability. Overall, these results show how human APC/C composition critically influences the cellular fate upon loss of SAC activity.