A relative shift in cloacal location repositions external genitalia in amniote evolution.

The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.

Venus trap in the mouse embryo reveals distinct molecular dynamics underlying specification of first embryonic lineages.

Mammalian development begins with the segregation of embryonic and extra-embryonic lineages in the blastocyst. Recent studies revealed cell-to-cell gene expression heterogeneity and dynamic cell rearrangements during mouse blastocyst formation. Thus, mechanistic understanding of lineage specification requires quantitative description of gene expression dynamics at a single-cell resolution in living embryos. However, only a few fluorescent gene expression reporter mice are available and quantitative live image analysis is limited so far. Here, we carried out a fluorescence gene-trap screen and established reporter mice expressing Venus specifically in the first lineages. Lineage tracking, quantitative gene expression and cell position analyses allowed us to build a comprehensive lineage map of mouse pre-implantation development. Our systematic analysis revealed that, contrary to the available models, the timing and mechanism of lineage specification may be distinct between the trophectoderm and the inner cell mass. While expression of our trophectoderm-specific lineage marker is upregulated in outside cells upon asymmetric divisions at 8- and 16-cell stages, the inside-specific upregulation of the inner-cell-mass marker only becomes evident at the 64-cell stage. This study thus provides a framework toward systems-level understanding of embryogenesis marked by high dynamicity and stochastic variability.

The Krüppel-associated box repressor domain can induce reversible heterochromatization of a mouse locus in vivo.

The study of chromatin and its regulators is key to understanding and manipulating transcription. We previously exploited the Krüppel-associated box (KRAB) transcriptional repressor domain, present in hundreds of vertebrate-specific zinc finger proteins, to assess the effect of its binding to gene bodies. These experiments revealed that the ectopic and doxycycline (dox)-controlled tet repressor KRAB fusion protein (tTRKRAB) can induce reversible and long-range silencing of cellular promoters. Here, we extend this system to in vivo applications and use tTRKRAB to achieve externally controllable repression of an endogenous mouse locus. We employed lentiviral-mediated transgenesis with promoterless TetO-containing gene traps to engineer a mouse line where the endogenous kinesin family member 2A (Kif2A) promoter drives a YFP reporter gene. When these mice were crossed to animals expressing the TetO-binding tTRKRAB repressor, this regulator was recruited to the Kif2A locus, and YFP expression was reduced. This effect was reversed when dox was given to embryos or adult mice, demonstrating that the cellular Kif2A promoter was only silenced upon repressor binding. Molecular analyses confirmed that tTRKRAB induced transcriptional repression through the spread of H3K9me3-containing heterochromatin, without DNA methylation of the trapped Kif2A promoter. Therefore, we demonstrate that targeting of tTRKRAB to a gene body in vivo results in reversible transcriptional repression through the spreading of facultative heterochromatin. This finding not only sheds light on KRAB-mediated transcriptional processes, but also suggests approaches for the externally controllable and reversible modulation of chromatin and transcription in vivo.

KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading.

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.

A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing.

BACKGROUND: KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences. METHOD: To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression. RESULTS: We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes. CONCLUSION: Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.

ARv7 Represses Tumor-Suppressor Genes in Castration-Resistant Prostate Cancer.

Androgen deprivation therapy for prostate cancer (PCa) benefits patients with early disease, but becomes ineffective as PCa progresses to a castration-resistant state (CRPC). Initially CRPC remains dependent on androgen receptor (AR) signaling, often through increased expression of full-length AR (ARfl) or expression of dominantly active splice variants such as ARv7. We show in ARv7-dependent CRPC models that ARv7 binds together with ARfl to repress transcription of a set of growth-suppressive genes. Expression of the ARv7-repressed targets and ARv7 protein expression are negatively correlated and predicts for outcome in PCa patients. Our results provide insights into the role of ARv7 in CRPC and define a set of potential biomarkers for tumors dependent on ARv7.

Role of steroid receptor and coregulator mutations in hormone-dependent cancers.

Steroid hormones mediate critical lineage-specific developmental and physiologic responses. They function by binding their cognate receptors, which are transcription factors that drive specific gene expression programs. The requirement of most prostate cancers for androgen and most breast cancers for estrogen has led to the development of endocrine therapies that block the action of these hormones in these tumors. While initial endocrine interventions are successful, resistance to therapy often arises. We will review how steroid receptor-dependent genomic signaling is affected by genetic alterations in endocrine therapy resistance. The detailed understanding of these interactions will not only provide improved treatment options to overcome resistance, but, in the future, will also be the basis for implementing precision cancer medicine approaches.

Enhancer-Mediated Oncogenic Function of the Menin Tumor Suppressor in Breast Cancer.

While the multiple endocrine neoplasia type 1 (MEN1) gene functions as a tumor suppressor in a variety of cancer types, we explored its oncogenic role in breast tumorigenesis. The MEN1 gene product menin is involved in H3K4 trimethylation and co-activates transcription. We integrated ChIP-seq and RNA-seq data to identify menin target genes. Our analysis revealed that menin-dependent target gene promoters display looping to distal enhancers that are bound by menin, FOXA1 and GATA3. In this fashion, MEN1 co-regulates a proliferative breast cancer-specific gene expression program in ER+ cells. In primary mammary cells, MEN1 exerts an anti-proliferative function by regulating a distinct expression signature. Our findings clarify the cell-type-specific functions of MEN1 and inform the development of menin-directed treatments for breast cancer.

PI3K/AKT Signaling Regulates H3K4 Methylation in Breast Cancer.

Post-translational histone H3 modifications regulate transcriptional competence. The mechanisms by which the epigenome is regulated in response to oncogenic signaling remain unclear. Here we show that H3K4me3 is increased in breast tumors driven by an activated PIK3CA allele and that inhibition of PI3K/AKT signaling reduces promoter-associated H3K4me3 in human breast cancer cells. We show that the H3K4 demethylase KDM5A is an AKT target and that phosphorylation of KDM5A regulates its nuclear localization and promoter occupancy. Supporting a role for KDM5A in mediating PI3K/AKT transcriptional effects, the decreased expression in response to AKT inhibition of a subset of cell-cycle genes associated with poor clinical outcome is blunted by KDM5A silencing. Our data identify a mechanism by which PI3K/AKT signaling modulates the cancer epigenome through controlling H3K4 methylation and suggest that KDM5A subcellular localization and genome occupancy may be pharmacodynamic markers of the activity of PI3K/AKT inhibitors currently in clinical development.

TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer.

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.

Prostate cancer. Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer.

Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer-associated mutations of SPOP, an E3 ubiquitin ligase substrate-binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, which implicated DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation.

EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent.

Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.

Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa.

Retinitis pigmentosa refers to a diverse group of hereditary diseases that lead to incurable blindness, affecting two million people worldwide. As a common pathology, rod photoreceptors die early, whereas light-insensitive, morphologically altered cone photoreceptors persist longer. It is unknown if these cones are accessible for therapeutic intervention. Here, we show that expression of archaebacterial halorhodopsin in light-insensitive cones can substitute for the native phototransduction cascade and restore light sensitivity in mouse models of retinitis pigmentosa. Resensitized photoreceptors activate all retinal cone pathways, drive sophisticated retinal circuit functions (including directional selectivity), activate cortical circuits, and mediate visually guided behaviors. Using human ex vivo retinas, we show that halorhodopsin can reactivate light-insensitive human photoreceptors. Finally, we identified blind patients with persisting, light-insensitive cones for potential halorhodopsin-based therapy.

KAP1-mediated epigenetic repression in the forebrain modulates behavioral vulnerability to stress.

KAP1 is an essential cofactor of KRAB-zinc finger proteins, a family of vertebrate-specific epigenetic repressors of largely unknown functions encoded in the hundreds by the mouse and human genomes. Here, we report that KAP1 is expressed at high levels and necessary for KRAB-mediated repression in mature neurons of the mouse brain. Mice deleted for KAP1 in the adult forebrain exhibit heightened levels of anxiety-like and exploratory activity and stress-induced alterations in spatial learning and memory. In the hippocampus, a small number of genes are dysregulated, including some imprinted genes. Chromatin analyses of the promoters of two genes markedly upregulated in knockout mice reveal decreased histone 3 K9-trimethylation and increased histone 3 and histone 4 acetylation. We propose a model in which the tethering of KAP1-associated chromatin remodeling factors via KRAB-ZFPs epigenetically controls gene expression in the hippocampus, thereby conditioning responses to behavioral stress.