In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these ''fragile'' nucleosomes play an important role in regulating RPG transcriptional output.

SummaryDNA methylation at imprinting control regions (ICRs) is established in gametes in a sex-specific manner and has to be stably maintained during development and in somatic cells to ensure the correct monoallelic expression of imprinted genes. In addition to DNA methylation, the ICRs are marked by allele-specific histone modifications. Whether these marks are essential for maintenance of genomic imprinting is largely unclear. Here, we show that the histone H3 lysine 9 methylases G9a and GLP are required for stable maintenance of imprinted DNA methylation in embryonic stem cells; however, their catalytic activity and the G9a/GLP-dependent H3K9me2 mark are completely dispensable for imprinting maintenance despite the genome-wide loss of non-imprinted DNA methylation in H3K9me2-depleted cells. We provide additional evidence that the G9a/GLP complex protects imprinted DNA methylation by recruitment of de novo DNA methyltransferases, which antagonize TET dioxygenass-dependent erosion of DNA methylation at ICRs.

Pioneer transcription factors (TFs) such as OCT4 can target silent genes embedded in nucleosome-dense regions. How nucleosome interaction enables TFs to target chromatin and determine cell identity remains elusive. Here, we systematically dissect OCT4 to show that nucleosome binding is encoded within the DNA-binding domain and yet can be uncoupled from free DNA binding. Furthermore, accelerating the binding kinetics of OCT4 to DNA enhances nucleosome binding. In cells, uncoupling nucleosome binding diminishes the ability of OCT4 to individually access closed chromatin, while more dynamic nucleosome binding results in expansive genome scanning within closed chromatin. However, both uncoupling and enhancing nucleosome binding are detrimental to inducing pluripotency from differentiated cells. Remarkably, stable interactions between OCT4 and nucleosomes are continuously required for maintaining the accessibility of pluripotency enhancers in stem cells. Our findings reveal how the affinity and residence time of OCT4-nucleosome complexes modulate chromatin accessibility during cell fate changes and maintenance. 3 MAIN To maintain cell identity, TFs are often associated with accessible enhancers and promoters of active genes. However, during cell fate changes, certain TFs target silent genes within closed chromatin, acting as "pioneer factors" 1 . For instance, Oct4, Sox2, Klf4 and c-Myc (OSKM) predominantly occupy open chromatin to maintain pluripotency in embryonic stem cells (ESCs) 2-4 . Yet, during early reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs), OSK, but not c-Myc, act as pioneer factors 5-9 .Nonetheless, binding of OSK to open chromatin has also been suggested to be important for reprogramming 10,11 . To date, it has not been possible to separate the conventional open chromatin binding from closed chromatin targeting, limiting the ability to directly examine the role of pioneer activity in pluripotency.We have previously shown that OSK can directly interact with nucleosomes like the paradigm pioneer factor FoxA, supporting a link between pioneer activity and nucleosome binding [12][13][14][15][16][17] . A systematic evolution of ligands by exponential enrichment (SELEX) study has revealed that a wide variety of DNA-binding domains (DBDs) can bind nucleosomes in vitro 18 . Commonly, DBDs containing short anchoring α helices have been shown to interact most strongly with nucleosomes in vitro 19 . However, FoxA-DBD requires an extra helical region to bind nucleosomes and open chromatin, suggesting that DBDs may not contain the full pioneer capacity 15,20 . Recently, the cryo-EM structure of OCT4-DBD co-bound with SOX2-DBD to an engineered nucleosome has shown a potential OCT4-SOX2 nucleosome readout that involves the OCT4 POU-specific domain (POUS) and SOX2 high mobility group (HMG), but not the POU-homeodomain (POUHD) of OCT4-DBD 21 . The cooperative interaction between OCT4 and SOX2 is critical for pluripotency