Cohesin is a well-known mediator of sister chromatid cohesion, but it also influences gene expression and development. These non-canonical roles of cohesin are not well understood, but are vital: gene expression and development are altered by modest changes in cohesin function that do not disrupt chromatid cohesion. To clarify cohesin's roles in transcription, we measured how cohesin controls RNA polymerase II (Pol II) activity by genome-wide chromatin immunoprecipitation and precision global run-on sequencing. On average, cohesin-binding genes have more transcriptionally active Pol II and promoter-proximal Pol II pausing than non-binding genes, and are more efficient, producing higher steady state levels of mRNA per transcribing Pol II complex. Cohesin depletion frequently decreases gene body transcription but increases pausing at cohesin-binding genes, indicating that cohesin often facilitates transition of paused Pol II to elongation. In many cases, this likely reflects a role for cohesin in transcriptional enhancer function. Strikingly, more than 95% of predicted extragenic enhancers bind cohesin, and cohesin depletion can reduce their association with Pol II, indicating that cohesin facilitates enhancer-promoter contact. Cohesin depletion decreases the levels of transcriptionally engaged Pol II at the promoters of most genes that don't bind cohesin, suggesting that cohesin controls expression of one or more broadly acting general transcription factors. The multiple transcriptional roles of cohesin revealed by these studies likely underlie the growth and developmental deficits caused by minor changes in cohesin activity.

Cohesin is crucial for proper chromosome segregation but also regulates gene transcription and organism development by poorly understood mechanisms. Using genome-wide assays in Drosophila developing wings and cultured cells, we find that cohesin functionally interacts with Polycomb group (PcG) silencing proteins at both silenced and active genes. Cohesin unexpectedly facilitates binding of Polycomb Repressive Complex 1 (PRC1) to many active genes, but their binding is mutually antagonistic at silenced genes. PRC1 depletion decreases phosphorylated RNA polymerase II and mRNA at many active genes but increases them at silenced genes. Depletion of cohesin reduces long-range interactions between Polycomb Response Elements in the invected-engrailed gene complex where it represses transcription. These studies reveal a previously unrecognized role for PRC1 in facilitating productive gene transcription and provide new insights into how cohesin and PRC1 control development.

Background: Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function. Results: We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function. Conclusions: Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.

44 Background: Several long noncoding RNAs (lncRNAs) have been shown to function as central 45 components of molecular machines that play fundamental roles in biology. While the number of annotated 46 lncRNAs in mammalian genomes has greatly expanded, their functions remain largely uncharacterized. 47This is compounded by the fact that identifying lncRNA loci that have robust and reproducible phenotypes 48 when mutated has been a challenge. 49Results: We previously generated a cohort of 20 lncRNA loci knockout mice. Here, we extend our initial 50 study and provide a more detailed analysis of the highly conserved lncRNA locus, Taurine Upregulated 51Gene 1 (Tug1). We report that Tug1 knockout male mice are sterile with complete penetrance due to a low 52 sperm count and abnormal sperm morphology. Having identified a lncRNA loci with a robust phenotype, we 53wanted to determine which, if any, potential elements contained in the Tug1 genomic region (DNA, RNA, 54protein, or the act of transcription) have activity. Using engineered mouse models and cell-based assays, 55we provide evidence that the Tug1 locus harbors three distinct regulatory activities -two noncoding and 56 one coding: (i) a cis DNA repressor that regulates many neighboring genes, (ii) a lncRNA that can regulate 57 genes by a trans-based function, and finally (iii) Tug1 encodes an evolutionary conserved peptide that when 58 overexpressed impacts mitochondrial membrane potential. 59Conclusions: Our results reveal an essential role for the Tug1 locus in male fertility and uncover three 60 distinct regulatory activities in the Tug1 locus, thus highlighting the complexity present at lncRNA loci. 61 62 63 KEYWORDS 64Tug1, lncRNA, fertility, DNA repressor, peptide, mouse, in vivo, RNA-seq

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