GAS6 receptor antagonism impairs adipocyte differentiation and adipose tissue development in mice (HRL, VC and IS)

ABSTRACT A low Mr receptor tyrosine kinase inhibitor (R428) with high affinity and selectivity for the GAS6 (growth arrest specific protein 6) receptor Axl was used to study a potential role of GAS6 signaling in adiposity. In vitro, R428 caused a concentration-dependent inhibition of preadipocyte differentiation into mature adipocytes, as evidenced by reduced lipid uptake. Inhibition of Axl mediated signaling was confirmed by reduced levels of phospho-Akt activity. In vivo, oral administration of R428 during 5 weeks to mice kept on high fat diet resulted in significantly reduced weight gain and subcutaneous (SC) and gonadal (GON) fat mass. This was associated with marked adipocyte hypotrophy, enhanced macrophage infiltration and apoptosis. Thus, affecting GAS6 signaling through receptor antagonism using a low Mr Axl antagonist impairs adipocyte differentiation and reduces adipose tissue development in a murine model of nutritionally induced obesity. JPET # 178046 -4

Germline HOXB13 mutations p.G84E and p.R217C do not confer an increased breast cancer risk

In breast cancer, high levels of homeobox protein Hox-B13 (HOXB13) have been associated with disease progression of ER-positive breast cancer patients and resistance to tamoxifen treatment. Since HOXB13 p.G84E is a prostate cancer risk allele, we evaluated the association between HOXB13 germline mutations and breast cancer risk in a previous study consisting of 3,270 familial non-BRCA1/2 breast cancer cases and 2,327 controls from the Netherlands. Although both recurrent HOXB13 mutations p.G84E and p.R217C were not associated with breast cancer risk, the risk estimation for p.R217C was not very precise. To provide more conclusive evidence regarding the role of HOXB13 in breast cancer susceptibility, we here evaluated the association between HOXB13 mutations and increased breast cancer risk within 81 studies of the international Breast Cancer Association Consortium containing 68,521 invasive breast cancer patients and 54,865 controls. Both HOXB13 p.G84E and p.R217C did not associate with the development of breast cancer in European women, neither in the overall analysis (OR = 1.035, 95% CI = 0.859-1.246, P = 0.718 and OR = 0.798, 95% CI = 0.482-1.322, P = 0.381 respectively), nor in specific high-risk subgroups or breast cancer subtypes. Thus, although involved in breast cancer progression, HOXB13 is not a material breast cancer susceptibility gene.Peer reviewe

Germline HOXB13 mutations p.G84E and p.R217C do not confer an increased breast cancer risk

Abstract: In breast cancer, high levels of homeobox protein Hox-B13 (HOXB13) have been associated with disease progression of ER-positive breast cancer patients and resistance to tamoxifen treatment. Since HOXB13 p.G84E is a prostate cancer risk allele, we evaluated the association between HOXB13 germline mutations and breast cancer risk in a previous study consisting of 3,270 familial non-BRCA1/2 breast cancer cases and 2,327 controls from the Netherlands. Although both recurrent HOXB13 mutations p.G84E and p.R217C were not associated with breast cancer risk, the risk estimation for p.R217C was not very precise. To provide more conclusive evidence regarding the role of HOXB13 in breast cancer susceptibility, we here evaluated the association between HOXB13 mutations and increased breast cancer risk within 81 studies of the international Breast Cancer Association Consortium containing 68,521 invasive breast cancer patients and 54,865 controls. Both HOXB13 p.G84E and p.R217C did not associate with the development of breast cancer in European women, neither in the overall analysis (OR = 1.035, 95% CI = 0.859–1.246, P = 0.718 and OR = 0.798, 95% CI = 0.482–1.322, P = 0.381 respectively), nor in specific high-risk subgroups or breast cancer subtypes. Thus, although involved in breast cancer progression, HOXB13 is not a material breast cancer susceptibility gene

Single cell profiling of an in vivo, RasV12 based tumour in the Drosophila eye primordium

The RasV12/scrib-/- tumour model has been widely used in Drosophila to study Ras-dependant oncogenesis and epithelial-mesenchymal transition (EMT). Recently, using a combination of ATAC-seq, FAIRE-seq and RNA-seq, we identified regulatory regions and two key transcription factors (AP-1 and Stat92E) involved in the development of these tumours. Using data from the whole tumour, it is impossible to determine whether these factors are part of the same gene regulatory networks (GRN) in a cell, or if they are controlling separate GRNs in different cells. In an attempt to answer this question, we used the C1 single cell auto-prep system (Fluidigm) and performed single-cell RNA-seq (SMART-seq2) and single-cell ATAC-seq on dissociated in vivo tumours. We envision that this approach will help answer this question and help enable us to start understand GRNs on the single cell level.status: publishe

Studying cellular differentiation in Drosophila using single cell ATAC-seq

Encoded within the genome is all of the information required to generate every cell type that exists in an organism, gene regulatory networks built from this information drive cellular differentiation from pluripotent cells to terminal differentiation. Although we are able to profile terminal cell states in many ways, it is currently unclear exactly how cells transition between states during differentiation. To unravel this transition, we aim to reverse engineer these gene regulatory networks and identify transcription factors and their targets which play a role in this process. To do this, we have taken advantage of single-cell ATAC-seq which allows us to profile the epigenome of individual cells. Particularly, we performed bulk and single-cell ATAC-seq across several hundreds of single cells, ranging from pluripotent progenitor cells to differentiating photoreceptor neurons in the Drosophila eye imaginal disc, as well as on RasV12/scrib-/- induced tumours, causing a block of differentiation in the same tissue. We compared the accessibility of various “cistromes” (sets of co-regulated enhancers) and identified meaningful cell clusters and possible developmental trajectories. The differentially accessible enhancers per cell cluster show motif enrichment for known transcription factors involved in the pluripotent state (e.g., Optix), the cancer-like cell state (e.g., AP-1, STAT92E), or the PR differentiation state (e.g., Tramtrack). In conclusion, scATAC-seq applied to in vivo tissue development allows us to identify changes in the chromatin landscape and infer regulatory programs of cellular differentiation at single cell resolution.status: accepte

Decoding epithelial enhancer logic using natural variation and single-cell ATAC-seq

Enhancers coordinate gene expression levels and the majority of disease related SNPs are located in these non-coding regions. Yet how genomic variation affects enhancer function is poorly understood. To investigate cis-regulatory variation in vivo, in an endogenous enhancer context, we profiled the genome-wide chromatin accessibility of epithelial tissues (imaginal discs), across 30 inbred Drosophila lines from the DGRP project. Statistical analysis identified 4289 chromatin accessibility QTLs (caQTL). We singled out the transcription factor Grainyhead as a key player, for which more than 50 caQTLs alter its binding site, thereby causing a concordant gain or loss in chromatin accesibility and in vivo enhancer activity. We further investigated the function and dynamics of Grainyhead bound enhancers using single-cell ATAC-seq on 322 cells, ranging from progenitors (high Grh), differentiated neurons (low Grh) and cells undergoing epithelial-to-mesenchymal transition (high to low Grh). We could construct developmental trajectories for these single-cells, driven by the changes in activity of the Grainyhead bound enhancers. Finally, to discriminate between functional and non-functional Grainyhead recognition motifs in the genome, we trained various machine learning algorithms and compared enhancers across different Drosophila species, allowing the identification of key enhancer features required for Grainyhead binding. In conclusion, we have directly linked non-coding SNPs to changes in enhancer activity and unveiled the basic regulatory logic of Grainyhead bound epithelial enhancers.status: publishe

EXPLORING THE P53 TRANSCRIPTIONAL NETWORK THROUGH INTEGRATIVE GENOMICS REVEALS NEW CANDIDATE TARGET GENES AND CO-FACTORS

As tumor suppressor many roles have been ascribed to p53 like cell cycle arrest and apoptosis but also metabolism and developmental processes. p53 functions as a transcription factor (TF) by interacting with a variety of target genes of which many have been reported but p53’s full targetome is likely incomplete. In addition, many other aspects of p53’s activity require further investigation. To address these questions we performed RNA-seq on MCF7-cells, revealing a list of differentially expressed genes. On this set we applied an in-house developed motif discovery tool called iRegulon, generating subsets of direct and indirect target genes. It also enabled us to retrieve possible master regulators like p53 itself but also possible new co-factors like AP-1. We observed E2F as regulator of the downregulated targets with a pronounced absence of the p53 motifs amongst these genes supporting the possibility of a p21-Rb-E2F approach for p53-repression. Next, we performed both ChIP-seq and FAIRE-seq in order to get a comprehensive view on the genomic landscape of p53 binding. While the p53 ChIP peaks improved our predicted set of p53 targets, the FAIRE profile established a correlation between open chromatin regions and upregulated genes. Finally we selected four enhancers from our direct targets for in vitro validation. Three of four enhancers showed the ability to functionally drive gene expression. In conclusion, by using NGS experiments, motif discovery and experimental validation we were able to address key questions about p53’s transcriptional mechanism and identify several new candidate target genes.status: accepte

Identification of Lineage-Specific Cis-Regulatory Modules Associated with Variation in Transcription Factor Binding and Chromatin Activity Using Ornstein-Uhlenbeck Models

Scoring the impact of noncoding variation on the function of cis-regulatory regions, on their chromatin state, and on the qualitative and quantitative expression levels of target genes is a fundamental problem in evolutionary genomics. A particular challenge is how to model the divergence of quantitative traits and to identify relationships between the changes across the different levels of the genome, the chromatin activity landscape, and the transcriptome. Here, we examine the use of the Ornstein-Uhlenbeck (OU) model to infer selection at the level of predicted cis-regulatory modules (CRMs), and link these with changes in transcription factor binding and chromatin activity. Using publicly available cross-species ChIP-Seq and STARR-Seq data we show how OU can be applied genome-wide to identify candidate transcription factors for which binding site and CRM turnover is correlated with changes in regulatory activity. Next, we profile open chromatin in the developing eye across three Drosophila species. We identify the recognition motifs of the chromatin remodelers, Trithorax-like and Grainyhead as mostly correlating with species-specific changes in open chromatin. In conclusion, we show in this study that CRM scores can be used as quantitative traits and that motif discovery approaches can be extended towards more complex models of divergence.status: publishe

Identification of lineage-specific Cis-regulatory modules associated with variation in transcription factor binding and chromatin activity using Ornstein-Uhlenbeck models

Scoring the impact of noncoding variation on the function of cis-regulatory regions, on their chromatin state, and on the qualitative and quantitative expression levels of target genes is a fundamental problem in evolutionary genomics. A particular challenge is how to model the divergence of quantitative traits and to identify relationships between the changes across the different levels of the genome, the chromatin activity landscape, and the transcriptome. Here, we examine the use of the Ornstein-Uhlenbeck (OU) model to infer selection at the level of predicted cis-regulatory-modules (CRMs), and link these with changes in transcription factor binding and chromatin activity. Using publicly available cross-species ChIP-Seq and STARR-Seq data we show how OU can be applied genome-wide to identify candidate transcription factors for which binding site and CRM turnover is correlated with changes in regulatory activity. Next, we profile open chromatin in the developing eye across three Drosophila species. We identify the recognition motifs of the chromatin remodelers, Trithorax-like and Grainyhead as mostly correlating with species-specific changes in open chromatin. In conclusion, we show in this study that CRM scores can be used as quantitative traits and that motif discovery approaches can be extended towards more complex models of divergence