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Abstract Hormonal contraception exposes women to synthetic progesterone receptor (PR) agonists, progestins, and transiently increases breast cancer risk. How progesterone and progestins affect the breast epithelium is poorly understood because we lack adequate models to study this. We hypothesized that individual progestins differentially affect breast epithelial cell proliferation and hence breast cancer risk. Using mouse mammary tissue ex vivo, we show that testosterone‐related progestins induce the PR target and mediator of PR signaling‐induced cell proliferation receptor activator of NF‐κB ligand (Rankl), whereas progestins with anti‐androgenic properties in reporter assays do not. We develop intraductal xenografts of human breast epithelial cells from 36 women, show they remain hormone‐responsive and that progesterone and the androgenic progestins, desogestrel, gestodene, and levonorgestrel, promote proliferation but the anti‐androgenic, chlormadinone, and cyproterone acetate, do not. Prolonged exposure to androgenic progestins elicits hyperproliferation with cytologic changes. Androgen receptor inhibition interferes with PR agonist‐ and levonorgestrel‐induced RANKL expression and reduces levonorgestrel‐driven cell proliferation. Thus, different progestins have distinct biological activities in the breast epithelium to be considered for more informed choices in hormonal contraception.

Abstract Invasive lobular carcinoma (ILC) is the most frequent special histological subtype of breast cancer, typically characterized by loss of E‐cadherin. It has clinical features distinct from other estrogen receptor‐positive (ER+) breast cancers but the molecular mechanisms underlying its characteristic biology are poorly understood because we lack experimental models to study them. Here, we recapitulate the human disease, including its metastatic pattern, by grafting ILC‐derived breast cancer cell lines, SUM‐44 PE and MDA‐MB‐134‐VI cells, into the mouse milk ducts. Using patient‐derived intraductal xenografts from lobular and non‐lobular ER+ HER2− tumors to compare global gene expression, we identify extracellular matrix modulation as a lobular carcinoma cell‐intrinsic trait. Analysis of TCGA patient datasets shows matrisome signature is enriched in lobular carcinomas with overexpression of elastin, collagens, and the collagen modifying enzyme LOXL1. Treatment with the pan LOX inhibitor BAPN and silencing of LOXL1 expression decrease tumor growth, invasion, and metastasis by disrupting ECM structure resulting in decreased ER signaling. We conclude that LOXL1 inhibition is a promising therapeutic strategy for ILC.

Abstract Background Positional weight matrix (PWM) is a de facto standard model to describe transcription factor (TF) DNA binding specificities. PWMs inferred from in vivo or in vitro data are stored in many databases and used in a plethora of biological applications. This calls for comprehensive benchmarking of public PWM models with large experimental reference sets. Results Here we report results from all-against-all benchmarking of PWM models for DNA binding sites of human TFs on a large compilation of in vitro (HT-SELEX, PBM) and in vivo (ChIP-seq) binding data. We observe that the best performing PWM for a given TF often belongs to another TF, usually from the same family. Occasionally, binding specificity is correlated with the structural class of the DNA binding domain, indicated by good cross-family performance measures. Benchmarking-based selection of family-representative motifs is more effective than motif clustering-based approaches. Overall, there is good agreement between in vitro and in vivo performance measures. However, for some in vivo experiments, the best performing PWM is assigned to an unrelated TF, indicating a binding mode involving protein-protein cooperativity. Conclusions In an all-against-all setting, we compute more than 18 million performance measure values for different PWM-experiment combinations and offer these results as a public resource to the research community. The benchmarking protocols are provided via a web interface and as docker images. The methods and results from this study may help others make better use of public TF specificity models, as well as public TF binding data sets.

Krüppel-associated box domain-zinc finger proteins (KRAB-ZFPs) are tetrapod-specific transcriptional repressors encoded in the hundreds by the human genome. In order to explore their as yet ill-defined impact on gene expression, we developed an ectopic repressor assay, allowing the study of KRAB-mediated transcriptional regulation at hundreds of different transcriptional units. By targeting a drug-controllable KRAB-containing repressor to gene-trapping lentiviral vectors, we demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases (kb) away from their DNA binding sites, with an efficiency which is generally higher for promoters located within 15 kb or less. Silenced promoters exhibit a loss of histone H3-acetylation, an increase in H3 lysine 9 trimethylation (H3K9me3), and a drop in RNA Pol II recruitment, consistent with a block of transcriptional initiation following the establishment of silencing marks. Furthermore, we reveal that KRAB-mediated repression is established by the long-range spreading of H3K9me3 and heterochromatin protein 1 beta (HP1beta) between the repressor binding site and the promoter. We confirm the biological relevance of this phenomenon by documenting KAP1-dependent transcriptional repression at an endogenous KRAB-ZFP gene cluster, where KAP1 binds to the 3' end of genes and mediates propagation of H3K9me3 and HP1beta towards their 5' end. Together, our data support a model in which KRAB/KAP1 recruitment induces long-range repression through the spread of heterochromatin. This finding not only suggests auto-regulatory mechanisms in the control of KRAB-ZFP gene clusters, but also provides important cues for interpreting future genome-wide DNA binding data of KRAB-ZFPs and KAP1.

The class II trans-activator CIITA is a transcriptional co-activator required for the expression of Major Histocompatibility Complex (MHC) genes. Although the latter function is well established, the global target-gene specificity of CIITA had not been defined. We therefore generated a comprehensive list of its target genes by performing genome-wide scans employing four different approaches designed to identify promoters that are occupied by CIITA in two key antigen presenting cells, B cells and dendritic cells. Surprisingly, in addition to MHC genes, only nine new targets were identified and validated by extensive functional and expression analysis. Seven of these genes are known or likely to function in processes contributing to MHC-mediated antigen presentation. The remaining two are of unknown function. CIITA is thus uniquely dedicated for genes implicated in antigen presentation. The finding that CIITA regulates such a highly focused gene expression module sets it apart from all other transcription factors, for which large-scale binding-site mapping has indicated that they exert pleiotropic functions and regulate large numbers of genes.