How evolutionary changes at enhancers affect the transcription of target genes remains an important open question. Previous comparative studies of gene expression have largely measured the abundance of mRNA, which is affected by post-transcriptional regulatory processes, hence limiting inferences about the mechanisms underlying expression differences. Here we directly measured nascent transcription in primate species, allowing us to separate transcription from post-transcriptional regulation. We used PRO-seq to map RNA polymerases in resting and activated CD4+ T-cells in multiple human, chimpanzee, and rhesus macaque individuals, with rodents as outgroups. We observed general conservation in coding and non-coding transcription, punctuated by numerous differences between species, particularly at distal enhancers and non-coding RNAs. Genes regulated by larger numbers of enhancers are more frequently transcribed at evolutionarily stable levels, despite reduced conservation at individual enhancers. Adaptive nucleotide substitutions are associated with lineage-specific transcription, and at one locus, SGPP2, we predict and experimentally validate that multiple substitutions contribute to human-specific transcription. Collectively, our findings suggest a pervasive role for evolutionary compensation across ensembles of enhancers that jointly regulate target genes.
The RET tyrosine kinase signaling pathway is involved in the development of endocrine resistant ER+ breast cancer. However, we know little about how ER+ cells activate RET signaling and initiate an endocrine resistant phenotype. Here we show that both ER+ endocrine resistant and sensitive breast cancers have a functional RET tyrosine kinase signaling pathway, but that endocrine sensitive breast cancer cells lack RET ligands that are necessary to drive endocrine resistance. Transcription of one RET ligand, GDNF, is necessary and sufficient to confer resistance in the ER+ MCF-7 cell line. Endogenous GDNF produced by endocrine resistant cells is translated, secreted into the media, and activates RET signaling in nearby cells. In patients, RET ligand expression predicts responsiveness to endocrine therapies and correlates with survival. Collectively, our findings show that ER+ tumor cells are “poised” for RET mediated endocrine resistance, expressing all components of the RET signaling pathway, but endocrine sensitive cells lack high expression of RET ligands that are necessary to initiate the resistance phenotype.
Background Peptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo. Methods Preliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice along with wild-type FVB/NJ (WT) and both strains of SKO mice for a range of reproductive defects. Results Controlled breeding trials found that male DKO mice appeared to take longer to have their first litter than WT controls. This tendency was maintained when these mice were mated to either DKO or WT females. Additionally, unsexed 2-day old DKO pups and male DKO weanlings both weighed significantly less than their WT counterparts, took significantly longer than WT males to reach puberty, and had consistently lower serum testosterone levels. Furthermore, 90-day old adult DKO males had smaller testes than WT males with increased rates of germ cell apoptosis. Conclusions The Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.
Background: Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression. Results: ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECMassociated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions. Conclusion: Outcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.
Discovering regulatory interactions between genes that specify the behavioral properties of cells remains an important challenge. We used the dynamics of transcriptional changes resolved by PRO-seq to identify a regulatory network responsible for endocrine resistance in breast cancer. We show that GDNF leads to endocrine resistance by switching the active state in a bi-stable feedback loop between GDNF, EGR1, and the master transcription factor ERα. GDNF stimulates MAP kinase, activating the transcription factors SRF and AP-1. SRF initiates an immediate transcriptional response, activating EGR1 and suppressing ERα. Newly translated EGR1 protein activates endogenous GDNF, leading to constitutive GDNF and EGR1 up-regulation, and the sustained down-regulation of ERα. Endocrine resistant MCF-7 cells are constitutively in the GDNF-high/ ERα-low state, suggesting that the state in the bi-stable feedback loop may provide a ‘memory’ of endocrine resistance. Thus, we identified a regulatory network switch that contributes to drug resistance in breast cancer.
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