Steroidogenic factor 1 (SF-1; Nr5a1) regulates the formation of the ovarian reserve.

The ovarian follicle reserve, formed pre- or perinatally, comprises all oocytes for lifetime reproduction. Depletion of this reserve results in infertility. Steroidogenic factor 1 (SF-1; Nr5a1) and liver receptor homolog 1 (LRH-1; Nr5a2) are two orphan nuclear receptors that regulate adult endocrine function, but their role in follicle formation is unknown. We developed models of conditional depletion of SF-1 or LRH-1 from prenatal ovaries. Depletion of SF-1, but not LRH-1, resulted in dramatically smaller ovaries and fewer primordial follicles. This was mediated by increased oocyte death, resulting from increased ovarian inflammation and increased Notch signaling. Major dysregulated genes were Iroquois homeobox 3 and 5 and their downstream targets involved in the establishment of the ovarian laminin matrix and oocyte-granulosa cell gap junctions. Disruptions of these pathways resulted in follicles with impaired basement membrane formation and compromised oocyte-granulosa communication networks, believed to render them more prone to atresia. This study identifies SF-1 as a key regulator of the formation of the ovarian reserve.

Newfound Coding Potential of Transcripts Unveils Missing Members of Human Protein Communities.

Recent proteogenomic approaches have led to the discovery that regions of the transcriptome previously annotated as non-coding regions [i.e., untranslated regions (UTRs), open reading frames overlapping annotated coding sequences in a different reading frame, and non-coding RNAs] frequently encode proteins, termed alternative proteins (altProts). This suggests that previously identified protein-protein interaction (PPI) networks are partially incomplete because altProts are not present in conventional protein databases. Here, we used the proteogenomic resource OpenProt and a combined spectrum- and peptide-centric analysis for the re-analysis of a high-throughput human network proteomics dataset thereby revealing the presence of 261 altProts in the network. We found 19 genes encoding both an annotated (reference) and an alternative protein interacting with each other. Of the 117 altProts encoded by pseudogenes, 38 are direct interactors of reference proteins encoded by their respective parental gene. Finally, we experimentally validate several interactions involving altProts. These data improve the blueprints of the human PPI network and suggest functional roles for hundreds of altProts.

UBB pseudogene 4 encodes functional ubiquitin variants.

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes UbKEKS (Q2K, K33E, Q49K, N60S). The functional consequences of UbKEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that UbKEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.

OpenProt: a more comprehensive guide to explore eukaryotic coding potential and proteomes.

Advances in proteomics and sequencing have highlighted many non-annotated open reading frames (ORFs) in eukaryotic genomes. Genome annotations, cornerstones of today's research, mostly rely on protein prior knowledge and on ab initio prediction algorithms. Such algorithms notably enforce an arbitrary criterion of one coding sequence (CDS) per transcript, leading to a substantial underestimation of the coding potential of eukaryotes. Here, we present OpenProt, the first database fully endorsing a polycistronic model of eukaryotic genomes to date. OpenProt contains all possible ORFs longer than 30 codons across 10 species, and cumulates supporting evidence such as protein conservation, translation and expression. OpenProt annotates all known proteins (RefProts), novel predicted isoforms (Isoforms) and novel predicted proteins from alternative ORFs (AltProts). It incorporates cutting-edge algorithms to evaluate protein orthology and re-interrogate publicly available ribosome profiling and mass spectrometry datasets, supporting the annotation of thousands of predicted ORFs. The constantly growing database currently cumulates evidence from 87 ribosome profiling and 114 mass spectrometry studies from several species, tissues and cell lines. All data is freely available and downloadable from a web platform (www.openprot.org) supporting a genome browser and advanced queries for each species. Thus, OpenProt enables a more comprehensive landscape of eukaryotic genomes' coding potential.

The Protein Coded by a Short Open Reading Frame, Not by the Annotated Coding Sequence, Is the Main Gene Product of the Dual-Coding Gene MIEF1.

Proteogenomics and ribosome profiling concurrently show that genes may code for both a large and one or more small proteins translated from annotated coding sequences (CDSs) and unannotated alternative open reading frames (named alternative ORFs or altORFs), respectively, but the stoichiometry between large and small proteins translated from a same gene is unknown. MIEF1, a gene recently identified as a dual-coding gene, harbors a CDS and a newly annotated and actively translated altORF located in the 5'UTR. Here, we use absolute quantification with stable isotope-labeled peptides and parallel reaction monitoring to determine levels of both proteins in two human cells lines and in human colon. We report that the main MIEF1 translational product is not the canonical 463 amino acid MiD51 protein but the small 70 amino acid alternative MiD51 protein (altMiD51). These results demonstrate the inadequacy of the single CDS concept and provide a strong argument for incorporating altORFs and small proteins in functional annotations.

Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins.

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins.

High-Resolution Genome-Wide Mapping of Nucleosome Positioning and Occupancy Level Using Paired-End Sequencing Technology.

Because of its profound influence on DNA accessibility for protein binding and thus on the regulation of diverse biological processes, nucleosome positioning has been studied for many years. In the past decade, high-throughput sequencing technologies have opened new perspectives in this research field by allowing the study of nucleosome positioning and occupancy on a genome-wide scale, therefore providing understanding on important aspects of chromatin packaging, as well as on various chromatin-template processes like transcription. In this chapter, we provide the protocol of MNase sequencing for the genome-wide mapping of nucleosomes using MNase to generate mononucleosomal DNA fragments and next-generation sequencing technology to identify their individual location.

Aggregate and Heatmap Representations of Genome-Wide Localization Data Using VAP, a Versatile Aggregate Profiler.

In the analysis of experimental data corresponding to the signal enrichment of chromatin features such as histone modifications throughout the genome, it is often useful to represent the signal over known regions of interest, such as genes, using aggregate or individual profiles. In the present chapter, we describe and explain the best practices on how to generate such profiles as well as other usages of the versatile aggregate profiler (VAP) tool (Coulombe et al., Nucleic Acids Res 42:W485-W493, 2014), with a particular focus on the new functionalities introduced in version 1.1.0 of VAP.

The histone variant H2A.Z is an important regulator of enhancer activity.

Gene regulatory programs in different cell types are largely defined through cell-specific enhancers activity. The histone variant H2A.Z has been shown to play important roles in transcription mainly by controlling proximal promoters, but its effect on enhancer functions remains unclear. Here, we demonstrate by genome-wide approaches that H2A.Z is present at a subset of active enhancers bound by the estrogen receptor alpha (ERα). We also determine that H2A.Z does not influence the local nucleosome positioning around ERα enhancers using ChIP sequencing at nucleosomal resolution and unsupervised pattern discovery. We further highlight that H2A.Z-enriched enhancers are associated with chromatin accessibility, H3K122ac enrichment and hypomethylated DNA. Moreover, upon estrogen stimulation, the enhancers occupied by H2A.Z produce enhancer RNAs (eRNAs), and recruit RNA polymerase II as well as RAD21, a member of the cohesin complex involved in chromatin interactions between enhancers and promoters. Importantly, their recruitment and eRNAs production are abolished by H2A.Z depletion, thereby revealing a novel functional link between H2A.Z occupancy and enhancer activity. Taken together, our findings suggest that H2A.Z acts as an important player for enhancer functions by establishing and maintaining a chromatin environment required for RNA polymerase II recruitment, eRNAs transcription and enhancer-promoters interactions, all essential attributes of enhancer activity.

VAP: a versatile aggregate profiler for efficient genome-wide data representation and discovery.

The analysis of genomic data such as ChIP-Seq usually involves representing the signal intensity level over genes or other genetic features. This is often illustrated as a curve (representing the aggregate profile of a group of genes) or as a heatmap (representing individual genes). However, no specific resource dedicated to easily generating such profiles is currently available. We therefore built the versatile aggregate profiler (VAP), designed to be used by experimental and computational biologists to generate profiles of genomic datasets over groups of regions of interest, using either an absolute or a relative method. Graphical representation of the results is automatically generated, and subgrouping can be performed easily, based on the orientation of the flanking annotations. The outputs include statistical measures to facilitate comparisons between groups or datasets. We show that, through its intuitive design and flexibility, VAP can help avoid misinterpretations of genomics data. VAP is highly efficient and designed to run on laptop computers by using a memory footprint control, but can also be easily compiled and run on servers. VAP is accessible at http://lab-jacques.recherche.usherbrooke.ca/vap/.

LRH-1 governs vital transcriptional programs in endocrine-sensitive and -resistant breast cancer cells.

Tumor characteristics are decisive in the determination of treatment strategy for patients with breast cancer. Patients with estrogen receptor α (ERα)-positive breast cancer can benefit from long-term hormonal treatment. Nonetheless, the majority of patients will develop resistance to these therapies. Here, we investigated the role of the nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in antiestrogen-sensitive and -resistant breast cancer cells. We identified genome-wide LRH-1-binding sites using ChIP-seq (chromatin immunoprecipitation sequencing), uncovering preferential binding to regions distal to transcriptional start sites. We further characterized these LRH-1-binding sites by integrating overlapping layers of specific chromatin marks, revealing that many LRH-1-binding sites are active and could be involved in long-range enhancer-promoter looping. Combined with transcriptome analysis of LRH-1-depleted cells, these results show that LRH-1 regulates specific subsets of genes involved in cell proliferation in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Furthermore, the LRH-1 transcriptional program is highly associated with a signature of poor outcome and high-grade breast cancer tumors in vivo. Herein, we report the genome-wide location and molecular function of LRH-1 in breast cancer cells and reveal its therapeutic potential for the treatment of breast cancers, notably for tumors resistant to treatments currently used in therapies.

Chromatin landscape and endocrine response in breast cancer.

Over two-thirds of breast cancers rely on estrogen receptor α (ERα) for their growth. Endocrine therapies antagonize estrogen-dependent ERα activation but resistance to these treatments occurs and is associated with poor prognosis. Crosstalk between alternative survival pathways and ERα are currently held as the primary cause of resistance. However, blocking these pathways does not cure endocrine therapy resistant breast cancer suggesting the existence of additional mechanisms. While cancer is commonly considered a genetic disease, the importance of epigenetic events in promoting tumor initiation and progression is increasingly recognized. Here, we consider how epigenetic modifications and alterations to the chromatin landscape contribute to endocrine therapy resistance by modulating ERα expression or altering its genomic activity.

Degradation of thyroid hormone receptor beta 1: existence of stable and unstable forms.

BACKGROUND: The degradation of many nuclear receptors is controlled by ligand-binding and mediated by the ubiquitin-proteasome pathway. However, the mechanisms implicated in thyroid hormone receptor (TR) degradation remain unclear. Our objective was to define the kinetics, mechanisms, and sub-cellular fractions involved in TRs degradation. METHODS: We used pulse-chase analyses, time-course experiments carried out in presence of cycloheximide (to inhibit new protein synthesis), and biochemical fractionation with Western blot analyses to determine the kinetics of the degradation of the TRß isoform, TRß1, in transiently transfected QBI-HEK 293A cells. RESULTS: We observed that TRß1 degradation is mediated by the proteasome pathway. Also, the kinetics of TRß1 degradation is atypical due to the co-existence of more than one TRß1 population, located in different cellular compartments and having different stability profiles. Moreover, TRß1 degradation was unaffected by a mutation in its putative PEST motif, which confers turnover of other proteins. CONCLUSION: Our findings introduce novel evidence suggesting that stable and unstable forms of TRß1, which might have distinct functions, co-exist in cells.

The effect of RanBPM on the regulation of the hypothalamic-pituitary axis by thyroid hormone receptors is isoform-specific.

Although crucial for TH homeostasis, the molecular mechanisms responsible of thyroid hormone receptors (TRs)-mediated regulation of the hypothalamic-pituitary-thyroid axis (HPT) axis remain unclear. We examined the role played by TR-isoforms in combination with RanBPM, a novel coactivator of TRs. In transient transfections studies with the human TRH and TSH-alpha subunit promoters, we found that the overexpression of RanBPM increases the transcriptional activity of all TR-isoforms by a magnitude of 1.7- to 3-fold. The addition of RanBPM, in the absence of THs, increased the ligand-independent activation (LIA) of TRalpha1 and TRbeta1 on both promoters tested by 300% and 200%, respectively, whereas, the LIA of TRbeta2 was not significantly modified. This data reinforces the concept of isoform-specific regulation of genes of the HPT axis and demonstrates that RanBPM may be an important factor to achieve adequate regulation of nTREs in the presence of low TH levels.