The aim of this paper is to describe a new regression strategy framework inspired on deep learning techniques for the NNPDF methodology [4]. The NNPDF methodology uses machine learning techniques in combination of Monte Carlo data generation to extract PDFs from experimental data. The NNPDF approach was pioneer in using artificial neural networks for the PDF parametrization and genetic algorithms for optimization. The NNPDF fitting framework has been constantly reviewed and upgraded in the last years, by including new features and methodological improvements which enhanced the quality of the released PDF sets [5,6,7]. Motivated by the new technologies and algorithms in use by the machine learning community, we dedicate this study to asses the impact of such new strategies in a modern PDF determination.
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Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.
Transcription factor (TF) binding to specific sites at transcription regulatory regions is a fundamental step in gene expression regulation. However, as the sequence of a given regulatory region does not change between different cell types, the regulation of cell-specific transcription is likely to depend on non-genetic regulators. TF access to regulatory elements is controlled by the chromatin structure, which in turn is modulated by epigenetic modifications, such as nucleosome remodelling, histone modifications and DNA methylation. Epigenetic modifications are generally classified into active or repressive according to their effect on gene expression. DNA tightly wrapped around nucleosomes due to repressive histone marks was shown to be refractory to TF binding1. Therefore, the activity of nucleosome remodellers that increase chromatin accessibility was deemed necessary prior to TF binding to compact chromatin2. Several studies identified a special class of TFs, called pioneer transcription factors (PFs), that access their target sites in condensed chromatin. Such an event increases chromatin accessibility via nucleosome remodelling, thus facilitating the recruitment of settler TFs that are, otherwise, unable to access condensed chromatin3,4,5,6. These findings hint for a more complex relationship between epigenetic- and genetic-based mechanisms in transcription regulation than previously thought. It is therefore important to establish whether and when epigenetic mechanisms constitute a primary event in the regulation of transcription and when do they simply result from the genetic composition of the regulatory regions (i.e. the presence of TF binding sites (TFBSs)).
Using a high-throughput explorative approach, our study methodically determined the ability of reported PFs to induce DNA demethylation at their binding sites in mouse embryonic stem cells (mESCs) and in vitro differentiated neuronal progenitors (NPs). Results show that, while many PFs do not affect the methylation status of their binding sites, a group of PFs that we call protective pioneer transcription factors (PPFs) prevent acquisition of DNA methylation, while another group called super pioneer transcription factors (SPFs) induce DNA demethylation at their methylated binding sites. Importantly, we show that, while most SPF-driven demethylation is Ten-Eleven Translocation (TET)-dependent, SOX2 (SRY (sex determining region Y)-box 2), an essential factor for the acquisition and maintenance of pluripotency12, inhibits DNA methyltransferase 1 (DNMT1)-dependent maintenance of methylation during replication. This inhibition is amplified by the co-binding of OCT4. Finally, while PFs are important to enhance chromatin accessibility, our results indicate that this may not be achieved solely by PF binding and the resulting demethylation, as exemplified by ATAC-Seq experiments around single SOX2 and CTCF binding sites. Interactions with multiple TFs are likely to be important for generating accessible chromatin.
Using Hi-TransMet, we identified PPFs that are able to protect against de novo methylation. Our screening both confirms previously reported PPFs (NRF118,57, CTCF and REST14) and identifies new ones, either constitutive (KLF4, SOX2, SOX9) or ESC- (KLF7, E2F1 and OTX2) and NP-specific (N-MYC) (Supplementary Table 3). Whether PPF binding shields its surrounding from DNA methyltransferases by steric hindrance or whether PPFs directly interact with DNMT3a/3b/3L leading to their inhibition awaits further studies. We also identified SPFs that, in addition to their known pioneering activities, can induce DNA demethylation at their binding sites. Constitutive SPFs are CTCF, REST, SOX2 and SOX17. ESC-specific SPFs are KLF4, E2F1, GR, N-MYC and SOX9, while NP-specific SPFs are FOXA1, FOXD3 and CREB (Supplementary Table 3). It should be noted that the cell specificity of PPFs and SPFs is largely driven by changes in their expression level, thus one could expect that these have the same effect in other cell types where they are expressed and active.
In June 2019, Kameny was one of the inaugural fifty American "pioneers, trailblazers, and heroes" inducted on the National LGBTQ Wall of Honor within the Stonewall National Monument (SNM) in New York City's Stonewall Inn.[58][59] The SNM is the first U.S. national monument dedicated to LGBTQ rights and history,[60] and the wall's unveiling was timed to take place during the 50th anniversary of the Stonewall riots.[61] 2ff7e9595c
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