Steroid receptors are involved in a wide array of cross talk mechanisms that regulate diverse biological processes, with significant implications in diseases, particularly in cancers. In prostate cancer, indirect cross talk between androgen receptor (AR) and glucocorticoid receptor NR3C1 (also known as GR) is well documented, wherein AR suppression by antiandrogen therapy leads to elevated GR levels, enabling GR to compensate for and replace AR signaling. However, the existence and impact of direct chromatin cross talk between AR and GR in prostate cancer have remained elusive. Here, our genome-wide investigations reveal that AR activation significantly expands GR chromatin binding. Mechanistically, AR induces remodeling of closed chromatin sites, facilitating GR binding to inaccessible sites. Importantly, coactivation of AR and GR results in distinct transcriptional responses at both the cell population and single-cell levels. Pathways affected by these transcriptional changes are generally associated with improved patient survival. Thus, the direct cross talk between AR and GR yields markedly different outcomes from the known role of GR in circumventing AR blockade by antiandrogens.

Glucocorticoids are a mainstay in the treatment of B-cell acute lymphoblastic leukemia (B-ALL). The glucocorticoid receptor (GR), a ligand-activated transcription factor (TF), mediates their actions. Chromatin occupancy, chromatin-protein networks (chromatomes) and gene programmes of GR are regulated by SUMOylation, a post-translational modification with therapeutic implications in other hematomalignancies. To unravel the GR-SUMOylation crosstalk in B-ALL, we induced hypoSUMOylation in NALM6 B-ALL cells with a SUMOylation inhibitor (SUMOi, ML-792). Genome-wide profiling of GR and SUMO chromatin-binding and chromatin accessibility revealed that hypoSUMOylation augmented GR chromatin occupancy and altered chromatin openness. Association with transcriptome data indicated that the hypoSUMOylation-induced GR-binding sites predominantly repressed genes associated with cell cycle and DNA replication. Consistently, hypoSUMOylation potentiated glucocorticoid-induced cell cycle arrest and growth suppression. Moreover, our proteomic analyses revealed that the protein network of chromatin-bound GR is tightly intertwined with SUMO2/3 and that SUMOylation modulates the stability of the network. The chromatome contained several B-cell TFs with cognate binding motifs found on GR-adjacent chromatin sites, indicating their simultaneous occupancy on chromatin. In sum, our data imply potential for targeting SUMOylation to increase sensitivity to glucocorticoids in B-ALL, supported by ex vivo data of glucocorticoid and SUMOi TAK-981 combination-treated B-ALL patient samples.

Exposure to metabolism-disrupting chemicals (MDCs), compounds largely belonging to the group of endocrine-disrupting chemicals (EDCs), is associated with metabolic dysfunctions such as dyslipidemia, insulin resistance and hepatic steatosis. Steroid hormone receptors (SHRs) are known targets for MDCs but their regulatory environment in the presence of environmental chemicals remains elusive. Here, we studied the activation and molecular interactions of SHRs exposed to 17 suspected MDCs including pesticides, plasticizers, pharmaceuticals, flame retardants, industrial chemicals and their metabolites by combining in vitro and in silico approaches. We first established and pre-validated reporter gene assays in HepG2 hepatoma cells to assess the activation of estrogen (ER), androgen (AR), glucocorticoid (GR) and progesterone (PR) receptors. Next, using RNA-seq and publicly available protein interaction data, we identified relevant SHR-interacting coregulators expressed in hepatic cells and measured their MDC-dependent interactions with SHRs using the Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) technology. Finally, we examined MDC binding to ER and GR using molecular dynamics simulations. These combined approaches lead to identification of MDCs capable of SHR activation at picomolar-to-low micromolar concentrations and paralleled with their ability to induce recruitment of multiple coregulators. MDCs induced distinct SHR-coregulator binding patterns involving multiple coactivators, corepressors and other modulatory proteins. Our results have broadened the test battery to detect MDCs and indicate that the activation of SHRs by MDCs is driven by diverse molecular interactions.

Steroid receptors are involved in a wide array of crosstalk mechanisms that regulate diverse biological processes, with significant implications in diseases, particularly in cancers. In prostate cancer, indirect crosstalk between androgen receptor (AR) and glucocorticoid receptor (GR) is well-documented, where GR replaces antiandrogen-inactivated AR becoming the disease driver. However, the existence and impact of direct chromatin crosstalk between AR and GR in prostate cancer have remained elusive. Our genome-wide investigations reveal that AR activation significantly expands GR chromatin binding. Mechanistically, AR induces remodeling of closed chromatin sites, facilitating GR binding to inaccessible sites. Importantly, coactivation of AR and GR results in distinct transcriptional responses at both the cell population and single-cell levels. Intriguingly, pathways affected by these transcriptional changes are generally associated with improved patient survival. Thus, the direct crosstalk between AR and GR yields markedly different outcomes from the known role of GR in circumventing AR blockade by antiandrogens. ### Competing Interest Statement The authors have declared no competing interest.

In sepsis, limited food intake and increased energy expenditure induce a starvation response, which is compromised by a quick decline in the expression of hepatic PPARα, a transcription factor essential in intracellular catabolism of free fatty acids. The mechanism upstream of this PPARα downregulation is unknown. We found that sepsis causes a progressive hepatic loss-of-function of HNF4α, which has a strong impact on the expression of several important nuclear receptors, including PPARα. HNF4α depletion in hepatocytes dramatically increases sepsis lethality, steatosis, and organ damage and prevents an adequate response to IL6, which is critical for liver regeneration and survival. An HNF4α agonist protects against sepsis at all levels, irrespectively of bacterial loads, suggesting HNF4α is crucial in tolerance to sepsis. In conclusion, hepatic HNF4α activity is decreased during sepsis, causing PPARα downregulation, metabolic problems, and a disturbed IL6-mediated acute phase response. The findings provide new insights and therapeutic options in sepsis.

The androgen receptor (AR) is pivotal in prostate cancer (PCa) progression and represents a critical therapeutic target. AR-mediated gene regulation involves intricate interactions with nuclear proteins, with many mediating and undergoing post-translational modifications that present alternative therapeutic avenues. Through chromatin proteomics in PCa cells, we identified SUMO ligases together with nuclear receptor coregulators and pioneer transcription factors within the AR's protein network. Intriguingly, this network displayed a significant association with SUMO2/3. To elucidate the influence of SUMOylation on AR chromatin interactions and subsequent gene regulation, we inhibited SUMOylation using ML-792 (SUMOi). While androgens generally facilitated the co-occupancy of SUMO2/3 and AR on chromatin, SUMOi induced divergent effects dependent on the type of AR-binding site (ARB). SUMOi augmented AR's pioneer-like binding on inaccessible chromatin regions abundant in androgen response elements (AREs) and diminished its interaction with accessible chromatin regions sparse in AREs yet rich in pioneer transcription factor motifs. The SUMOi-impacted ARBs divergently influenced AR-regulated genes; those associated with AR-mediated activation played roles in negative regulation of cell proliferation, while those with AR-mediated repression were involved in pattern formation. In conclusion, our findings underscore the pervasive influence of SUMOylation in shaping AR's role in PCa cells, potentially unveiling new therapeutic strategies.

Treatment of prostate cancer relies predominantly on the inhibition of androgen receptor (AR) signaling. Despite the initial effectiveness of the antiandrogen therapies, the cancer often develops resistance to the AR blockade. One mechanism of the resistance is glucocorticoid receptor (GR)-mediated replacement of AR function. Nevertheless, the mechanistic ways and means how the GR-mediated antiandrogen resistance occurs have remained elusive. Here, we have discovered several crucial features of GR action in prostate cancer cells through genome-wide techniques. We detected that the replacement of AR by GR in enzalutamide-exposed prostate cancer cells occurs almost exclusively at pre-accessible chromatin sites displaying FOXA1 occupancy. Counterintuitively to the classical pioneer factor model, silencing of FOXA1 potentiated the chromatin binding and transcriptional activity of GR. This was attributed to FOXA1-mediated repression of the NR3C1 (gene encoding GR) expression via the corepressor TLE3. Moreover, the small-molecule inhibition of coactivator p300's enzymatic activity efficiently restricted GR-mediated gene regulation and cell proliferation. Overall, we identified chromatin pre-accessibility and FOXA1-mediated repression as important regulators of GR action in prostate cancer, pointing out new avenues to oppose steroid receptor-mediated antiandrogen resistance.

A zebrafish orthologue (whnb) of the mouse nude gene is expressed in the epithelial compartment of the embryonic thymic rudiment 179 Stage-and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis 187 D. Engelkamp (Germany) Cloning of three mouse Unc5 genes and their expression patterns at mid-gestation 191 Expression of Sox8, Sox9 and Sox10 in the developing valves and autonomic nerves of the embryonic heart 199 Molecular cloning and expression pattern of a Cubitus interruptus homologue from the mulberry silkworm Bombyx mori 203 F. Matsushita, T. Kameyama, T. Marunouchi (Japan) NZF-2b is a novel predominant form of mouse NZF-2/MyT1, expressed in differentiated neurons especially at higher levels in newly generated ones 209 R.A. Udani, K.M. Karrer (USA) ASI1, a gene encoding a novel leucine zipper protein, is induced during development of the macronucleus in Tetrahymena 215 Expression of the LIM-homeodomain gene Lmx1a (dreher) during development of the mouse nervous system 225 Oocytes and embryos of Xenopus laevis express two different isoforms of germ cell nuclear factor (GCNF, NR6A1) 261 C.M.H. Vacalla, T. Theil (Germany) Cst, a novel mouse gene related to Drosophila Castor, exhibits dynamic expression patterns during neurogenesis and heart development 265

The androgen receptor (AR) plays a central role in the development and maintenance of the male phenotype. The binding of androgens to the receptor induces interactions between the carboxyterminal ligand-binding domain and the highly conserved 23FQNLF27 motif in the aminoterminal domain. The role of these so-called N/C interactions in AR functioning is debated. In vitro assays show that mutating the AR in the 23FQNLF27 motif (called ARNoC) attenuates the AR transactivation of reporter genes, has no effect on ligand binding, but does affect protein-protein interactions with several AR coregulators. To test the in vivo relevance of the N/C interaction, we analyzed the consequences of the genomic introduction of the ARNoC mutation in mice. Surprisingly, the ARNoC/Y mice show a normal male development, with unaffected male anogenital distance and normal accessory sex glands, male circulating androgen levels, body composition, and fertility. The responsiveness of androgen target genes in kidney, prostate, and testes was also unaffected. We thus conclude that the N/C interactions in the AR are not essential for the development of a male phenotype under normal physiological conditions.

Abstract The androgen receptor (AR) plays a crucial role in the development and maintenance of the male phenotype, as shown in patients with androgen insensitivity syndrome (AIS). The AR is a nuclear receptor that needs to homodimerize to execute its role as transcription factor. Dimerization can occur through three different modes: via the DNA-binding domain (DBD), via the ligand-binding domain and via an interaction between the LBD and the aminoterminal domain. Dimerization via the DBD is very well known and occurs through the D-box located in the second zinc finger. DBD dimerization is generally accepted for all steroid receptors, but some of them show that D-box mutations do not inactivate them completely. However, for the AR, no monomeric transactivation capacity was described yet. To study the role of D-box independent activity of the AR, we introduced a double point mutation in the D-box that is predicted to disrupt DBD dimerization (ARDmon). These two point mutations were found in separate patients suffering from partial AIS. In vitro studies with this mutant suggest that the remaining activity depends on the androgen response element under investigation. We show that ligand binding was not affected by the D-box mutation. Furthermore, BioID assays indicated no changes in coregulatory interactions. When introducing the corresponding mutation in mice, this led to a phenotype that is intermediate between global ARKO and wild type (WT) males. Based on their anogenital distance (AGD) and the absence of nipples at early age, the ARDmon/Y mice were identified as males. Follow-up of their AGD and body weight, however, revealed clear differences with both WT males and females. Furthermore, hypospadias was observed in the ARDmon/Y mice. Testes were smaller in size compared to the WT males and first analysis showed almost no expression of well-known AR regulated genes. Reminiscent structures of the epididymis were found. In summary, the ARDmon mouse model shows an intermediate AIS phenotype. Via orchidectomy in combination with androgen replacement therapy, we will now investigate remaining androgen responses in the ARDmon/Y mice and elucidate response elements on which the AR can act in a D-box independent way. Additionally, we generated the first rodent PAIS model with a genetic cause of hypospadias. Presentation: Saturday, June 11, 2022 12:45 p.m. - 1:00 p.m.

In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.

Steroid receptors (SRs) constitute an important class of signal-dependent transcription factors (TFs). They regulate a variety of key biological processes and are crucial drug targets in many disease states. In particular, estrogen (ER) and androgen receptors (AR) drive the development and progression of breast and prostate cancer, respectively. Thus, they represent the main specific drug targets in these diseases. Recent evidence has suggested that the crosstalk between signal-dependent TFs is an important step in the reprogramming of chromatin sites; a signal-activated TF can expand or restrict the chromatin binding of another TF. This crosstalk can rewire gene programs and thus alter biological processes and influence the progression of disease. Lately, it has been postulated that there may be an important crosstalk between the AR and the ER with other SRs. Especially, progesterone (PR) and glucocorticoid receptor (GR) can reprogram chromatin binding of ER and gene programs in breast cancer cells. Furthermore, GR can take the place of AR in antiandrogen-resistant prostate cancer cells. Here, we review the current knowledge of the crosstalk between SRs in breast and prostate cancers. We emphasize how the activity of ER and AR on chromatin can be modulated by other SRs on a genome-wide scale. We also highlight the knowledge gaps in the interplay of SRs and their complex interactions with other signaling pathways and suggest how to experimentally fill in these gaps.

Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4’s functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.

The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K →R ) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K →R or Zfp451-/- ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog The differentiation defect of Satb2K →R ESCs correlates with altered higher-order chromatin interactions relative to Satb2wt ESCs. Upon RA treatment of Satb2wt ESCs, SATB2 interacts with ZFP451 and the LSD1/CoREST complex and gains binding at differentiation genes, which is not observed in RA-treated Satb2K →R cells. Thus, SATB2 SUMOylation may contribute to the rewiring of transcriptional networks and the chromatin interactome of ESCs in the transition of pluripotency to differentiation.

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance.

Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.

Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.

Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (ARLmon/Y). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in ARLmon/Y mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation in vivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition. This study reveals the contribution of ligand-binding domain (LBD) dimerization to androgen receptor (AR) activity. Disrupting LBD dimerization affects multiple receptor functions, proposing this interface as new therapeutic target. This study reveals the contribution of ligand-binding domain (LBD) dimerization to androgen receptor (AR) activity. Disrupting LBD dimerization affects multiple receptor functions, proposing this interface as new therapeutic target.

Ovarian deficiency, including premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR), represents one of the main causes of female infertility. POI is a genetically heterogeneous condition but current understanding of its genetic basis is far from complete, with the cause remaining unknown in the majority of patients. The genes that regulate DOR have been reported but the genetic basis of DOR has not been explored in depth. Both conditions are likely to lie along a continuum of degrees of decrease in ovarian reserve. We performed genomic analysis via whole exome sequencing (WES) followed by in silico analyses and functional experiments to investigate the genetic cause of ovarian deficiency in ten affected women. We achieved diagnoses for three of them, including the identification of novel variants in STAG3, GDF9, and FANCM. We identified potentially causative FSHR variants in another patient. This is the second report of biallelic GDF9 and FANCM variants, and, combined with functional support, validates these genes as bone fide autosomal recessive "POI genes". We also identified new candidate genes, NRIP1, XPO1, and MACF1. These genes have been linked to ovarian function in mouse, pig, and zebrafish respectively, but never in humans. In the case of NRIP1, we provide functional support for the deleterious nature of the variant via SUMOylation and luciferase/β-galactosidase reporter assays. Our study provides multiple insights into the genetic basis of POI/DOR. We have further elucidated the involvement of GDF9, FANCM, STAG3 and FSHR in POI pathogenesis, and propose new candidate genes, NRIP1, XPO1, and MACF1, which should be the focus of future studies.