Efficient enrichment of synchronized mouse spermatocytes suitable for genome-wide analysis.

Chromatin-based mechanisms regulating developmental transitions during meiosis are fundamental but understudied aspects of male gametogenesis. Indeed, chromatin undergoes extensive remodeling dur-ing meiosis, leading to specific patterns of gene expression and chromosome organization, which ulti-mately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin binding sites by ChIP-seq. High quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin binding sites.

Doxorubicin Impacts the Chromatin Binding of Hmgb1, Histone H1 and Retinoic Acid Receptor

Doxorubicin (Dox), a widely used anticancer DNA-binding drug, affects chromatin in multiple ways, and these effects contribute to both its efficacy and dose-limiting side-effects, especially cardiotoxicity. Here we studied the Dox effects on the chromatin binding of the architectural proteins high mobility group B1 (HMGB1) and the linker histone H1, and the transcription factor retinoic acid receptor (RARα) by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS), in live cells. At lower drug concentrations, Dox increased the binding of HMGB1 to DNA while decreasing the binding of the linker histone H1. At higher doses that correspond to the peak plasma concentrations reached in chemotherapy, Dox reduced the binding of HMGB1 as well. This biphasic effect is interpreted in terms of a hierarchy of competition between the ligands involved and Dox-induced local conformational changes of nucleosome-free DNA. When combined, FRAP and FCS mobility data suggest that Dox decreases the overall binding of RARα to DNA, an effect that was only partially overcome by agonist binding. The intertwined interactions described likely contribute to the effects as well as side-effects of Dox.

MDM2-Driven Ubiquitination Rapidly Removes p53 from Its Cognate Promoters

MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53–chromatin interactions.

HMGA1 Has Predictive Value in Response to Chemotherapy in Gastric Cancer

Gastric cancer is a serious health problem worldwide. Although its incidence is decreasing, the five-year survival rate remains low. Thus, it is essential to identify new biomarkers that could promote better diagnosis and treatment of patients with gastric cancer. High-mobility group AT-hook 1 (HMGA1) is a non-histone, chromatin-binding protein that has been found overexpressed in several tumor types. It has been correlated with invasion, metastasis, and drug resistance, leading to worse patient survival. The aim of this work was to evaluate the clinical value of HMGA1 in gastric cancer. HMGA1 expression was analyzed by immunohistochemistry in a single hospital series (n = 323) of gastric adenocarcinoma cases (stages I to IV) with clinicopathological and treatment data. In this series, HMGA1 expression showed no significant relevance as a prognostic biomarker. Nevertheless, a significantly better overall survival was observed in cases with high levels of HMGA1 when they were treated with chemotherapy, compared to the nontreated ones, implying that they can benefit more from treatment than patients with low expression of HMGA1. We thereby show for the first time that HMGA1 expression has a substantial value as a biomarker of response to chemotherapy in gastric cancer.

Cistrome And Transcriptome Analysis Identifies Unique Androgen Receptor (AR) And AR- V7 Splice Variant Chromatin Binding And Transcriptional Activities

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

Differential Histone-DNA Interactions Dictate Nucleosome Recognition of the Pioneer Transcription Factor Sox

Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin for stimulating vital cellular processes. Expanding on the recent findings, we aim to unravel the universal binding mode of the famous Sox PTF. Our findings show that the base specific hydrogen bonding (base reading) and the local DNA changes (shape reading) are required for sequence-specific nucleosomal DNA recognition by Sox. Among different nucleosomal positions, base and shape reading can be satisfied at super helical location 2 (SHL2). This indicates that due to distinct histone-DNA interactions, SHL2 acts transparently to Sox binding, where SHL4 permits solely shape reading, and SHL0 (dyad) allows no reading. We also show that at SHL2, Sox binds to its recognition sequence without imposing any major conformational changes, if its consensus DNA sequence is located at the solvent-facing nucleosomal DNA strand. These data explain how Sox have evolved to perfectly adapt for chromatin binding.

Cistrome and transcriptome analysis identifies unique androgen receptor (AR) and AR- V7 splice variant chromatin binding and transcriptional activities

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

Androgen receptor and MYC equilibration centralizes on developmental super-enhancer

Androgen receptor (AR) in prostate cancer (PCa) can drive transcriptional repression of multiple genes including MYC, and supraphysiological androgen is effective in some patients. Here, we show that this repression is independent of AR chromatin binding and driven by coactivator redistribution, and through chromatin conformation capture methods show disruption of the interaction between the MYC super-enhancer within the PCAT1 gene and the MYC promoter. Conversely, androgen deprivation in vitro and in vivo increases MYC expression. In parallel, global AR activity is suppressed by MYC overexpression, consistent with coactivator redistribution. These suppressive effects of AR and MYC are mitigated at shared AR/MYC binding sites, which also have markedly higher levels of H3K27 acetylation, indicating enrichment for functional enhancers. These findings demonstrate an intricate balance between AR and MYC, and indicate that increased MYC in response to androgen deprivation contributes to castration-resistant PCa, while decreased MYC may contribute to responses to supraphysiological androgen therapy.

Asgard ESCRT-III and VPS4 reveal evolutionary conserved chromatin binding properties of the ESCRT machinery

The ESCRT machinery drive membrane remodeling in numerous processes in eukaryotes. Genes encoding for ESCRT proteins have been identified in Asgard archaea, a newly discovered superphylum, currently recognized as the ancestor of all eukaryotes. This begs the question of the functional evolutionary origin of this machinery and its conservation across lineages. Here, we find that Asgard-ESCRTs exhibit conserved DNA-binding properties, which is derived from recruitment of specific members. We show that Asgard-ESCRT-III/VPS4 homologs interact with one another inside mammalian cells, associate with chromatin, and recruit their counterparts to organize in discrete foci in the mammalian nucleus. This is congruent with human-ESCRT-III homologs. We find that human- and Asgard-ESCRT-IIIs associate with chromatin via the same N terminal domain, and that human-ESCRT-III can recruit Asgard-VPS4 to the nucleus to form foci. Therefore, ESCRTs possess chromatin binding properties that were preserved through the billion years of evolution that separate Asgard and human cells.

A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer

Mutations in the PHIP/BRWD2 chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations in PHIP expression are linked to cancer. Precisely how PHIP functions in these contexts is not fully understood. Here we demonstrate that PHIP is a chromatin-associated CRL4 ubiquitin ligase substrate receptor and is required for CRL4 recruitment to chromatin. PHIP binds to chromatin through a trivalent reader domain consisting of a H3K4-methyl binding Tudor domain and two bromodomains (BD1 and BD2). Using semisynthetic nucleosomes with defined histone post-translational modifications, we characterize PHIPs BD1 and BD2 as respective readers of H3K14ac and H4K12ac, and identify human disease-associated mutations in each domain and the intervening linker region that likely disrupt chromatin binding. These findings provide new insight into the biological function of this enigmatic chromatin protein and set the stage for the identification of both upstream chromatin modifiers and downstream targets of PHIP in human disease.