A Urine-based DNA Methylation Marker Test to Detect Upper Tract Urothelial Carcinoma: A Prospective Cohort Study.

PURPOSE: We explored the accuracy of a urine-based epigenetic test for detecting upper tract urothelial carcinoma. MATERIALS AND METHODS: Under an Institutional Review Board-approved protocol, urine samples were prospectively collected from primary upper tract urothelial carcinoma patients before radical nephroureterectomy, ureterectomy, or ureteroscopy between December 2019 and March 2022. Samples were analyzed with Bladder CARE, a urine-based test that measures the methylation levels of 3 cancer biomarkers (TRNA-Cys, SIM2, and NKX1-1) and 2 internal control loci using methylation-sensitive restriction enzymes coupled with quantitative polymerase chain reaction. Results were reported as the Bladder CARE Index score and quantitatively categorized as positive (>5), high risk (2.5-5), or negative (<2.5). The findings were compared with those of 1:1 sex/age-matched cancer-free healthy individuals. RESULTS: Fifty patients (40 radical nephroureterectomy, 7 ureterectomy, and 3 ureteroscopy) with a median (IQR) age of 72 (64-79) years were included. Bladder CARE Index results were positive in 47, high risk in 1, and negative in 2 patients. A significant correlation was found between Bladder CARE Index values and tumor size. Urine cytology was available for 35 patients, of whom 22 (63%) results were false-negative. Upper tract urothelial carcinoma patients had significantly higher Bladder CARE Index values compared to the controls (mean 189.3 vs 1.6, P < .001). The sensitivity, specificity, positive predictive value, and negative predictive value of the Bladder CARE test for detecting upper tract urothelial carcinoma were 96%, 88%, 89%, and 96%, respectively.Conclusions:Bladder CARE is an accurate urine-based epigenetic test for the diagnosis of upper tract urothelial carcinoma, with much higher sensitivity than standard urine cytology.

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity.

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.

ATP-dependent chromatin remodeling factors and their roles in affecting nucleosome fiber composition.

ATP-dependent chromatin remodeling factors of the SNF2 family are key components of the cellular machineries that shape and regulate chromatin structure and function. Members of this group of proteins have broad and heterogeneous functions ranging from controlling gene activity, facilitating DNA damage repair, promoting homologous recombination to maintaining genomic stability. Several chromatin remodeling factors are critical components of nucleosome assembly processes, and recent reports have identified specific functions of distinct chromatin remodeling factors in the assembly of variant histones into chromatin. In this review we will discuss the specific roles of ATP-dependent chromatin remodeling factors in determining nucleosome composition and, thus, chromatin fiber properties.

Clinical evaluation of Bladder CARE, a new epigenetic test for bladder cancer detection in urine samples.

BACKGROUND: Bladder cancer (BC) is the 5th most common cancer in the USA. Non-muscle invasive bladder cancer represents about 70% of all cases and has generally a favorable outcome. However, recurrence rates as high as 60 to 70% and progression rates of 10 to 20% necessitate intensive surveillance with cystoscopy. The invasiveness and high cost of cystoscopy poses significant burden on BC patients as well as on the healthcare system. In this study we test the feasibility of a simple, sensitive, and non-invasive detection of BC using Bladder CARE test in urine samples. RESULTS: Urine from 136 healthy and 77 BC subjects was collected using the at-home Bladder CARE Urine Collection Kit and analyzed with Bladder CARE test. The test measures the methylation level of three BC-specific biomarkers and two internal controls using methylation-sensitive restriction enzymes coupled with qPCR. Bladder CARE showed an overall sensitivity of 93.5%, a specificity of 92.6%, and a PPV and NPV of 87.8% and 96.2%, respectively. Bladder CARE has an LOD as low as 0.046%, which equates to detecting 1 cancer cell for every 2,200 cells analyzed. We also provided evidence that bisulfite-free methods to assess DNA methylation, like Bladder CARE, are advantageous compared to conventional methods that rely on bisulfite conversion of the DNA. CONCLUSION: Highly sensitive detection of BC in urine samples is possible using Bladder CARE. The low LOD of the test and the measurement of epigenetic biomarkers make Bladder CARE a good candidate for the early detection of BC and possibly for the routine screening and surveillance of BC patients. Bladder CARE and the at-home urine sample collection system have the potential to (1) reduce unnecessary invasive testing for BC (2) reduce the burden of surveillance on patients and on the healthcare system, (3) improve the detection of early stage BC, and (4) allow physicians to streamline the monitoring of patients.

Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates.

The study of RNA dynamics, specifically RNA transcription and decay rates, has gained increasing attention in recent years because various mechanisms have been discovered that affect mRNA half-life, thereby ultimately controlling protein output. Therefore, there is a need for methods enabling minimally invasive, simple and high-throughput determination of RNA stability that can be applied to determine RNA transcription and decay rates in cells and organisms. We have recently developed a protocol which we named TUC-seq to directly distinguish newly synthesized transcripts from the preexisting pool of transcripts by metabolic labeling of nascent RNAs with 4-thiouridine (4sU) followed by osmium tetroxide-mediated conversion of 4sU to cytidine (C) and direct sequencing. In contrast to other related methods (SLAM-seq, TimeLapse-seq), TUC-seq converts 4sU to a native C instead of an alkylated or otherwise modified nucleoside derivative. TUC-seq can be applied to any cell type that is amenable to 4sU labeling. By employing different labeling strategies (pulse or pulse-chase labeling), it is suitable for a broad field of applications and provides a fast and highly efficient means to determine mRNA transcription and decay rates.

Role for Chromatin Remodeling Factor Chd1 in Learning and Memory.

Precise temporal and spatial regulation of gene expression in the brain is a prerequisite for cognitive processes such as learning and memory. Epigenetic mechanisms that modulate the chromatin structure have emerged as important regulators in this context. While posttranslational modification of histones or the modification of DNA bases have been examined in detail in many studies, the role of ATP-dependent chromatin remodeling factors (ChRFs) in learning- and memory-associated gene regulation has largely remained obscure. Here we present data that implicate the highly conserved chromatin assembly and remodeling factor Chd1 in memory formation and the control of immediate early gene (IEG) response in the hippocampus. We used various paradigms to assess short-and long-term memory in mice bearing a mutated Chd1 gene that gives rise to an N-terminally truncated protein. Our data demonstrate that the Chd1 mutation negatively affects long-term object recognition and short- and long-term spatial memory. We found that Chd1 regulates hippocampal expression of the IEG early growth response 1 (Egr1) and activity-regulated cytoskeleton-associated (Arc) but not cFos and brain derived neurotrophic factor (Bdnf), because the Chd1-mutation led to dysregulation of Egr1 and Arc expression in naive mice and in mice analyzed at different stages of object location memory (OLM) testing. Of note, Chd1 likely regulates Egr1 in a direct manner, because chromatin immunoprecipitation (ChIP) assays revealed enrichment of Chd1 upon stimulation at the Egr1 genomic locus in the hippocampus and in cultured cells. Together these data support a role for Chd1 as a critical regulator of molecular mechanisms governing memory-related processes, and they show that this function involves the N-terminal serine-rich region of the protein.

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications.

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.

Impaired Contextual Fear Extinction Learning is Associated with Aberrant Regulation of CHD-Type Chromatin Remodeling Factors.

Successful attenuation of fearful memories is a cognitive process requiring initiation of highly coordinated transcription programs. Chromatin-modulating mechanisms such as DNA methylation and histone modifications, including acetylation, are key regulators of these processes. However, knowledge concerning the role of ATP-dependent chromatin remodeling factors (ChRFs) being required for successful fear extinction is lacking. Underscoring the potential importance of these factors that alter histone-DNA contacts within nucleosomes are recent genome-wide association studies linking several ChRFs to various human cognitive and psychiatric disorders. To better understand the role of ChRFs in the brain, and since to date little is known about ChRF expression in the brain, we performed a comprehensive survey of expression levels of 24 ATP-dependent remodelers across different brain areas, and we identified several distinct high molecular weight complexes by chromatographic methods. We next aimed to gain novel insight into the potential regulation of ChRFs in different brain regions in association with normal and impaired fear extinction learning. To this end, we established the 129S1/SvImJ (S1) laboratory mouse strain as a model for compromised contextual fear extinction learning that can be rescued by dietary zinc restriction (ZnR). Using this model along with genetically related but fear extinction-competent 129S6/SvEv (S6) mice as controls, we found that impaired fear extinction in S1 was associated with enhanced ventral hippocampal expression of CHD1 and reduced expression of CHD5 that was normalized following successful rescue of impaired fear extinction. Moreover, a select reduction in CHD3 expression was observed in the ventral hippocampus (vHC) following successful rescue of fear extinction in S1 mice. Taken together, these data provide novel insight into the regulation of specific ChRFs following an impaired cognitive process and its rescue, and they suggest that imbalance of CHD-type remodeler levels, which consequently may lead to changes of transcriptional programs, may be an underlying mechanism involved in impaired fear extinction learning and its therapeutic rescue.

Embryonic stem cell differentiation requires full length Chd1.

The modulation of chromatin dynamics by ATP-dependent chromatin remodeling factors has been recognized as an important mechanism to regulate the balancing of self-renewal and pluripotency in embryonic stem cells (ESCs). Here we have studied the effects of a partial deletion of the gene encoding the chromatin remodeling factor Chd1 that generates an N-terminally truncated version of Chd1 in mouse ESCs in vitro as well as in vivo. We found that a previously uncharacterized serine-rich region (SRR) at the N-terminus is not required for chromatin assembly activity of Chd1 but that it is subject to phosphorylation. Expression of Chd1 lacking this region in ESCs resulted in aberrant differentiation properties of these cells. The self-renewal capacity and ESC chromatin structure, however, were not affected. Notably, we found that newly established ESCs derived from Chd1(Δ2/Δ2) mutant mice exhibited similar differentiation defects as in vitro generated mutant ESCs, even though the N-terminal truncation of Chd1 was fully compatible with embryogenesis and post-natal life in the mouse. These results underscore the importance of Chd1 for the regulation of pluripotency in ESCs and provide evidence for a hitherto unrecognized critical role of the phosphorylated N-terminal SRR for full functionality of Chd1.

CenH3/CID incorporation is not dependent on the chromatin assembly factor CHD1 in Drosophila.

CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histone H3.3 in the paternal pronucleus as well as in transcriptionally active nuclei in Drosophila embryos. The S. pombe and vertebrate orthologs of CHD1 have been implicated in the assembly of the centromeric histone H3 variant CenH3(CENP-A), which occurs in a DNA replication-independent manner. Here, we examined whether CHD1 participates in the assembly of CenH3(CID) in Drosophila. In contrast to the findings in fission yeast and vertebrate cells, our evidence clearly argues against such a role for CHD1 in Drosophila. CHD1 does not localize to centromeres in either S2 cells or developing fly embryos. Down-regulation of CHD1 in S2 cells by RNAi reveals unchanged levels of CenH3(CID) at the centromeres. Most notably, ablation of functional CHD1 in Chd1 mutant fly embryos does not interfere with centromere and kinetochore assembly, as the levels and localization of CenH3(CID), CENP-C and BubR1 in the mutant embryos remain similar to those seen in wild-type embryos. These results indicate that Drosophila CHD1 has no direct function in the incorporation of the centromeric H3 variant CenH3(CID) into chromatin. Therefore, centromeric chromatin assembly may involve different mechanisms in different organisms.

Chromatin and DNA modifications in the Opaque2-mediated regulation of gene transcription during maize endosperm development.

The maize (Zea mays) Opaque2 (O2) gene encodes an endosperm-specific bZIP-type transcription activator. In this study, we analyzed O2 targets for chromatin and DNA modifications and transcription factors binding during endosperm development and in leaves. In leaves, O2 targets exhibit high cytosine methylation levels and transcriptionally silent chromatin, enriched with histones H3 dimethylated at Lys-9 (H3K9me2) and Lys-27 (H3K27me2). Transcriptional activation in the endosperm occurs through a two-step process, with an early potentiated state and a later activated state. The potentiated state has cytosine demethylation at symmetric sites, substitution of H3K9me2 and H3K27me2 with histones H3 acetylated at Lys-14 (H3K14ac) and dimethylated at Lys-4 (H3K4me2), and increased DNaseI sensitivity. During the activated state, the mRNA of O2 targets accumulates in correspondence to RNPII, O2, and Ada2/Gcn5 coactivator binding. The active state also exhibits further increases of H3K14ac/H3K4me2 and DNaseI accessibility levels and deposition of histone H3 acetylated at Lys-9 and trimethylated at Lys-4. Analysis of o2 mutants revealed that O2 targets differ in their dependence on O2 activity for coactivator recruitment and for formation of specific chromatin modification profiles. These results indicate gene-specific involvement of mechanisms that modify chromatin states in the O2-mediated regulation of transcription.