Marks Of Active Transcription Research Articles

Abstract 2290: Integration of cell-free DNA end motifs and fragment lengths can improve identification of active genes in liquid biopsies

Abstract Multiple studies have shown that the plasma cell-free DNA (cfDNA) from cancer patients differs from healthy individuals in both fragment length and fragment end motifs (FEMs). Yet, there is a lack of understanding regarding how the two factors combined can be associated with cancer and gene transcription. In this study, we evaluated cfDNA fragmentomics in plasma from lung cancer patients (n = 17) and healthy individuals (n = 7) using targeted sequencing (lung cancer: n = 12, healthy: n = 7) and whole-genome sequencing (WGS) (lung cancer: n = 5). A personal gene expression profile was established with H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq). cfChIP-seq isolates nucleosomes with histone marks of active transcription directly from plasma, meaning cfDNA enrichment with cfChIP reflects gene activity in the cells of origin. Short cfDNA fragments were isolated using in vitro size-selection. Somatic mutations were identified from input cfDNA through the AVENIO pipeline. The findings were validated in an external public dataset comprising of WGS-cfDNA data from healthy individuals. Cancer patients contain a larger fraction of short (<150bp) cfDNA fragments compared to healthy individuals. Cancer patients also demonstrate an enrichment of distinct FEMs compared to healthy individuals. Investigating the association between gene activity and fragment lengths revealed that genes with high expression display an enrichment of short cfDNA fragments (median = 19.99%, IQR: 16.94% - 27.13%, P < 0.0001) compared to genes with low expression. Furthermore, distinct GC-rich FEMs are enriched in active genes. Combining the frequency of short cfDNA fragments with the presence of distinct FEMs results in a further enrichment of the highest expressed genes (median = 37.85%, IQR: 30.10% - 39.49%, P < 0.0001). In vitro size selection of short cfDNA also results in enrichment of GC-rich FEMs. In vitro size-selection can isolate cfDNA representing active genes and in vitro size-selection enrichment correlates with the cfChIP-seq enrichment (Spearman’s ρ range: 0.499-0.882). This study expands the knowledge regarding cfDNA fragmentomics. We document how cancer patients and healthy individuals have different fragmentomic features. In addition, we shed new light on how gene activity in the cells of origin is associated with cfDNA fragment lengths and FEMs. Together, the results can increase the utility of liquid biopsies by determining gene activity in cancer patients. Citation Format: Christoffer Trier Maansson, Louise Skov Thomsen, Peter Meldgaard, Anders Lade Nielsen, Boe Sandahl Sorensen. Integration of cell-free DNA end motifs and fragment lengths can improve identification of active genes in liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2290.

Dynamic DNA N 6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus.

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N 6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

Polyglutamine-expanded ATXN7 alters a specific epigenetic signature underlying photoreceptor identity gene expression in SCA7 mouse retinopathy

BackgroundSpinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder that primarily affects the cerebellum and retina. SCA7 is caused by a polyglutamine expansion in the ATXN7 protein, a subunit of the transcriptional coactivator SAGA that acetylates histone H3 to deposit narrow H3K9ac mark at DNA regulatory elements of active genes. Defective histone acetylation has been presented as a possible cause for gene deregulation in SCA7 mouse models. However, the topography of acetylation defects at the whole genome level and its relationship to changes in gene expression remain to be determined.MethodsWe performed deep RNA-sequencing and chromatin immunoprecipitation coupled to high-throughput sequencing to examine the genome-wide correlation between gene deregulation and alteration of the active transcription marks, e.g. SAGA-related H3K9ac, CBP-related H3K27ac and RNA polymerase II (RNAPII), in a SCA7 mouse retinopathy model.ResultsOur analyses revealed that active transcription marks are reduced at most gene promoters in SCA7 retina, while a limited number of genes show changes in expression. We found that SCA7 retinopathy is caused by preferential downregulation of hundreds of highly expressed genes that define morphological and physiological identities of mature photoreceptors. We further uncovered that these photoreceptor genes harbor unusually broad H3K9ac profiles spanning the entire gene bodies and have a low RNAPII pausing. This broad H3K9ac signature co-occurs with other features that delineate superenhancers, including broad H3K27ac, binding sites for photoreceptor specific transcription factors and expression of enhancer-related non-coding RNAs (eRNAs). In SCA7 retina, downregulated photoreceptor genes show decreased H3K9 and H3K27 acetylation and eRNA expression as well as increased RNAPII pausing, suggesting that superenhancer-related features are altered.ConclusionsOur study thus provides evidence that distinctive epigenetic configurations underlying high expression of cell-type specific genes are preferentially impaired in SCA7, resulting in a defect in the maintenance of identity features of mature photoreceptors. Our results also suggest that continuous SAGA-driven acetylation plays a role in preserving post-mitotic neuronal identity.

Epigenetic regulator Cfp1 safeguards male meiotic progression by regulating meiotic gene expression

Meiosis occurs specifically in germ cells to produce sperm and oocytes that are competent for sexual reproduction. Multiple factors are required for successful meiotic entry, progression, and termination. Among them, trimethylation of histone H3 on lysine 4 (H3K4me3), a mark of active transcription, has been implicated in spermatogenesis by forming double-strand breaks (DSBs). However, the role of H3K4me in transcriptional regulation during meiosis remains poorly understood. Here, we reveal that mouse CXXC finger protein 1 (Cfp1), a component of the H3K4 methyltransferase Setd1a/b, is dynamically expressed in differentiating male germ cells and safeguards meiosis by controlling gene expression. Genetic ablation of mouse CFP1 in male germ cells caused complete infertility with failure in prophase I of the 1st meiosis. Mechanistically, CFP1 binds to genes essential for spermatogenesis, and its loss leads to a reduction in H3K4me3 levels and gene expression. Importantly, CFP1 is highly enriched within the promoter/TSS of target genes to elevate H3K4me3 levels and gene expression at the pachytene stage of meiotic prophase I. The most enriched genes were associated with meiosis and homologous recombination during the differentiation of spermatocytes to round spermatids. Therefore, our study establishes a mechanistic link between CFP1-mediated transcriptional control and meiotic progression and might provide an unprecedented genetic basis for understanding human sterility.

Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis

Nucleophosmin (NPM1) is a multifunctional histone chaperone that can activate acetylation‐dependent transcription from chromatin templates in vitro. Acetylation of NPM1 by p300 has been shown to further enhance its transcription activation potential. Moreover, its total and acetylated pools are increased in oral squamous cell carcinoma. However, the role of NPM1 or its acetylated form (AcNPM1) in transcriptional regulation in cells and oral tumorigenesis is not fully elucidated. Using ChIP‐seq analyses, we provide the first genome‐wide profile of AcNPM1 and show that AcNPM1 is enriched at transcriptional regulatory elements. AcNPM1 co‐occupies marks of active transcription at promoters and DNase I hypersensitive sites at enhancers. In addition, using a high‐throughput protein interaction profiling approach, we show that NPM1 interacts with RNA Pol II, general transcription factors, mediator subunits, histone acetyltransferase complexes, and chromatin remodelers. NPM1 histone chaperone activity also contributes to its transcription activation potential. Further, NPM1 depletion leads to decreased AcNPM1 occupancy and reduced expression of genes required for proliferative, migratory and invasive potential of oral cancer cells. NPM1 depletion also abrogates the growth of orthotopic tumors in mice. Collectively, these results establish that AcNPM1 functions as a coactivator during during RNA polymerase II‐driven transcription and regulates the expression of genes that promote oral tumorigenesis.

The histone H3-lysine 4-methyltransferase Mll4 regulates the development of growth hormone-releasing hormone-producing neurons in the mouse hypothalamus

In humans, inactivating mutations in MLL4, which encodes a histone H3-lysine 4-methyltransferase, lead to Kabuki syndrome (KS). While dwarfism is a cardinal feature of KS, the underlying etiology remains unclear. Here we report that Mll4 regulates the development of growth hormone-releasing hormone (GHRH)-producing neurons in the mouse hypothalamus. Our two Mll4 mutant mouse models exhibit dwarfism phenotype and impairment of the developmental programs for GHRH-neurons. Our ChIP-seq analysis reveals that, in the developing mouse hypothalamus, Mll4 interacts with the transcription factor Nrf1 to trigger the expression of GHRH-neuronal genes. Interestingly, the deficiency of Mll4 results in a marked reduction of histone marks of active transcription, while treatment with the histone deacetylase inhibitor AR-42 rescues the histone mark signature and restores GHRH-neuronal production in Mll4 mutant mice. Our results suggest that the developmental dysregulation of Mll4-directed epigenetic control of transcription plays a role in the development of GHRH-neurons and dwarfism phenotype in mice.

Modification of the Histone Landscape with JAK Inhibition in Myeloproliferative Neoplasms.

Simple SummaryThe introduction of JAK inhibitors has provided objective benefits to many patients with myeloproliferative neoplasms. However, the overall efficacy of these drugs is limited and improved therapeutic options are required. Epigenetic dysregulation is increasingly observed to play a role in these disorders. This study evaluates the changing landscape of histone modifications in myeloproliferative neoplasm (MPN) cell line models and patient samples from the MAJIC clinical trial following administration of the JAK inhibitor ruxolitinib, demonstrating that ruxolitinib has an epigenetic effect. This changing histone modification landscape may offer a target for further therapy to augment treatment.Dysregulation of epigenetic processes is increasingly understood to play a role in the pathogenesis of myeloproliferative neoplasms (MPNs). Ruxolitinib, a JAK/STAT inhibitor, has proved a useful addition to the therapeutic arsenal for these disorders, but has limited disease modifying activity. We determined the effect of JAK inhibition on the histone landscape of MPN cells in cell line models of MPNs and validated using samples from the MAJIC randomised clinical trial of ruxolitinib in polycythaemia vera and essential thrombocythaemia. We demonstrated an epigenetic modifying effect of ruxolitinib using a histone modification assay. The majority of 21 histone H3 modifications were upregulated, with H3K27me3 and H3K36me2 significant in the combined cell line results. Chromatin immunoprecipitation and sequencing (CHIP-seq) for three marks of interest, H3K4me1, H3K4me3 and H3K27ac, was consistent with the histone modification assay showing a significant increase in H3K4me3 and H3K27ac peaks at promoter regions, both marks of active transcription. In contrast, RNA sequencing demonstrates a coordinated reduction in gene expression in a number of cell pathways including PI3K-AKT signalling, transcriptional misregulation in cancer and JAK-STAT signalling in spite of these histone changes. This highlights the complex mechanisms of transcriptional control within the cells which was reflected in analysis of the histone landscape in patient samples following ruxolitinib treatment.

Broad domains of histone H3 lysine 4 trimethylation in transcriptional regulation and disease.

Histone modifications affect transcription by changing the chromatin structure. In particular, histone H3 lysine 4 trimethylation (H3K4me3) is one of the most recognized epigenetic marks of active transcription. While many studies have provided evidence of the correlation between H3K4me3 and active transcription, details regarding the mechanism involved remain unclear. The first study on the broad H3K4me3 domain was reported in 2014; subsequently, the function of this domain has been studied in various cell types. In this review, we summarized the recent studies on the role of the broad H3K4me3 domain in transcription, development, memory formation, and several diseases, including cancer and autoimmune diseases. The broadest H3K4me3 domains are associated with increased transcriptional precision of cell-type-specific genes related to cell identity and other essential functions. The broad H3K4me3 domain regulates maternal zygotic activation in early mammalian development. In systemic autoimmune diseases, high expression of immune-responsive genes requires the presence of the broad H3K4me3 domain in the promoter-proximal regions. Transcriptional repression of tumor-suppressor genes is associated with the shortening of the broad H3K4me3 domains in cancer cells. Additionally, the broad H3K4me3 domain interacts with the super-enhancer to regulate cancer-associated genes. During memory formation, H3K4me3 breadth is regulated in the hippocampus CA1 neurons. Taken together, these findings indicate that H3K4me3 breadth is essential for the regulation of the transcriptional output across multiple cell types.

Quercetin and its derivative Q2 modulate chromatin dynamics in adipogenesis and Q2 prevents obesity and metabolic disorders in rats

Recently the attention of the scientific community has focused on the ability of polyphenols to counteract adverse epigenetic regulation involved in the development of complex conditions such as obesity. The aim of this study was to investigate the epigenetic mechanisms underlying the anti-adiposity effect of Quercetin (3,3′,4′,5,7-pentahydroxyflavone) and of one of its derivatives, Q2 in which the OH groups have been replaced by acetyl groups. In 3 T3-L1 preadipocytes, Quercetin and Q2 treatment induce chromatin remodeling and histone modifications at the 5′ regulatory region of the two main adipogenic genes, c/EBPα and PPARγ. Chromatin immunoprecipitation assays revealed a concomitant increase of histone H3 di-methylation at Lys9, a typical mark of repressed gene promoters, and a decrease of histone H3 di-methylation at Lys 4, a mark of active transcription. At the same time, both compounds inhibited histone demethylase LSD1 recruitment to the 5′ region of c/EBPα and PPARγ genes, a necessary step for adipogenesis. The final effect is a significant reduction in c/EBPα and PPARγ gene expression and attenuated adipogenesis. Q2 supplementation in rats reduced the gain in body weight and in white adipose tissue, as well as the increase in adipocyte size determined by high fat diet. Moreover, Q2 improved dyslipidemia, glucose tolerance and decreased the hepatic lipid accumulation by activating the expression of beta-oxidation related genes. Our data suggest that Q2, as well as Quercetin, has the potential to revert the unfavorable epigenomic profiles associated with obesity onset. This opens the possibility to use these compounds in targeted prevention strategies against obesity.

Effects of culture condition on epigenomic profiles of brain tumor cells.

Personalized cancer medicine offers the promise of more effective treatments that are tailored to an individual's own dynamic cancer phenotype. Meanwhile, tissue-engineering approaches to modeling tumors may complement these advances by providing a powerful new approach to understanding the adaptation dynamics occurring during treatment. However, in both of these areas new tools will be required to gain a full picture of the genetic and epigenetic regulators of phenotype dynamics occurring in the small populations of cells that drive resistance. In this study, we perform epigenomic analysis of brain tumor cells that are collected from micro-engineered three-dimensional tumor models, overcoming the challenges associated with the small numbers of cells contained within these micro-tissue niches, in this case collecting ~1,000 cells per sample. Specifically, we use a high-resolution epigenomic analysis method known as microfluidic-oscillatory-washing-based chromatin immunoprecipitation with sequencing (MOWChIP-seq) to analyze histone methylation patterns (H3K4me3). We identified gene loci that are associated with the H3K4me3 modification, which is generally a mark of active transcription. We compared methylation patterns in standard 2D cultures and 3D cultures based on type I collagen hydrogels, under both normoxic and hypoxic conditions. We found that culture dimensionality drastically impacted the H3k4me3 profile and resulted in differential modifications in response to hypoxic stress. Differentially H3K4me3-marked regions under the culture conditions used in this study have important implications for gene expression differences that have been previously observed. In total, our work illustrates a direct connection between cell culture or tissue niche condition and genome-wide alterations in histone modifications, providing the first steps towards analyzing the spatiotemporal variations in epigenetic regulation of cancer cell phenotypes. This study, to our knowledge, also represents the first time broad-spectrum epigenomic analysis has been applied to small cell samples collected from engineered micro-tissues.

SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

The life cycle of HPV is tied to the differentiation status of its host cell, with productive replication, late gene expression and virion production restricted to the uppermost layers of the stratified epithelium. HPV DNA is histone-associated, exhibiting a chromatin structure similar to that of the host chromosome. Although HPV chromatin is subject to histone post-translational modifications, how the viral life cycle is epigenetically regulated is not well understood. SETD2 is a histone methyltransferase that places the trimethyl mark on H3K36 (H3K36me3), a mark of active transcription. Here, we define a role for SETD2 and H3K36me3 in the viral life cycle. We have found that HPV positive cells exhibit increased levels of SETD2, with SETD2 depletion leading to defects in productive viral replication and splicing of late viral RNAs. Reducing H3K36me3 by overexpression of KDM4A, an H3K36me3 demethylase, or an H3.3K36M transgene also blocks productive viral replication, indicating a significant role for this histone modification in facilitating viral processes. H3K36me3 is enriched on the 3’ end of the early region of the high-risk HPV31 genome in a SETD2-dependent manner, suggesting that SETD2 may regulate the viral life cycle through the recruitment of H3K36me3 readers to viral DNA. Intriguingly, we have found that activation of the ATM DNA damage kinase, which is required for productive viral replication, is necessary for the maintenance of H3K36me3 on viral chromatin and for processing of late viral RNAs. Additionally, we have found that the HPV31 E7 protein maintains the increased SETD2 levels in infected cells through an extension of protein half-life. Collectively, our findings highlight the importance of epigenetic modifications in driving the viral life cycle and identify a novel role for E7 as well as the DNA damage response in the regulation of viral processes through epigenetic modifications.

Dual Src Kinase/Pretubulin Inhibitor KX-01, Sensitizes ERα-negative Breast Cancers to Tamoxifen through ERα Reexpression.

Unlike breast cancer that is positive for estrogen receptor-α (ERα), there are no targeted therapies for triple-negative breast cancer (TNBC). ERα is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERα expression in TNBC may sensitize patients to endocrine therapy. Expression of c-Src and ERα are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to reexpression of ERα in TNBC. KX-01 is a peptide substrate-targeted Src/pretubulin inhibitor in clinical trials for solid tumors. KX-01 (1 mg/kg body weight-twice daily) inhibited growth of tamoxifen-resistant MDA-MB-231 and MDA-MB-157 TNBC xenografts in nude mice that was correlated with Src kinase inhibition. KX-01 also increased ERα mRNA and protein, as well as increased the ERα targets progesterone receptor (PR), pS2 (TFF1), cyclin D1 (CCND1), and c-myc (MYC) in MDA-MB-231 and MDA-MB-468, but not MDA-MB-157 xenografts. MDA-MB-231 and MDA-MB-468 tumors exhibited reduction in mesenchymal markers (vimentin, β-catenin) and increase in epithelial marker (E-cadherin) suggesting mesenchymal-to-epithelial transition (MET). KX-01 sensitized MDA-MB-231 and MDA-MB-468 tumors to tamoxifen growth inhibition and tamoxifen repression of the ERα targets pS2, cyclin D1, and c-myc. Chromatin immunoprecipitation (ChIP) of the ERα promoter in KX-01-treated tumors demonstrated enrichment of active transcription marks (acetyl-H3, acetyl-H3Lys9), dissociation of HDAC1, and recruitment of RNA polymerase II. Methylation-specific PCR and bisulfite sequencing demonstrated no alteration in ERα promoter methylation by KX-01. These data demonstrate that in addition to Src kinase inhibition, peptidomimetic KX-01 restores ERα expression in TNBC through changes in histone acetylation that sensitize tumors to tamoxifen.Implications: Src kinase/pretubulin inhibitor KX-01 restores functional ERα expression in ERα- breast tumors, a novel treatment strategy to treat triple-negative breast cancer. Mol Cancer Res; 15(11); 1491-502. ©2017 AACR.

ING3 promotes prostate cancer growth by activating the androgen receptor

BackgroundThe androgen receptor (AR) is a major driver of prostate cancer, and increased AR levels and co-activators of the receptor promote the development of prostate cancer. INhibitor of Growth (ING) proteins target lysine acetyltransferase or lysine deacetylase complexes to the histone H3K4Me3 mark of active transcription, to affect chromatin structure and gene expression. ING3 is a stoichiometric member of the TIP60 lysine acetyltransferase complex implicated in prostate cancer development.MethodsBiopsies of 265 patients with prostate cancer were stained for ING3, pan-cytokeratin, and DNA. LNCaP and C4-2 androgen-responsive cells were used for in vitro assays including immunoprecipitation, western blotting, Luciferase reporter assay and quantitative polymerase chain reaction. Cell viability and migration assays were performed in prostate cancer cell lines using scrambled siRNA or siRNA targeting ING3.ResultsWe find that ING3 levels and AR activity positively correlate in prostate cancer. ING3 potentiates androgen effects, increasing expression of androgen-regulated genes and androgen response element-driven reporters to promote growth and anchorage-independent growth. Conversely, ING3 knockdown inhibits prostate cancer cell growth and invasion. ING3 activates the AR by serving as a scaffold to increase interaction between TIP60 and the AR in the cytoplasm, enhancing receptor acetylation and translocation to the nucleus. Activation is independent of ING3's ability to target the TIP60 complex to H3K4Me3, identifying a previously unknown chromatin-independent cytoplasmic activity for ING3. In agreement with in vitro observations, analysis of The Cancer Genome Atlas (TCGA) data (n = 498) and a prostate cancer tissue microarray (n = 256) show that ING3 levels are higher in aggressive prostate cancers, with high levels of ING3 predicting shorter patient survival in a low AR subgroup. Including ING3 levels with currently used indicators such as the Gleason score provides more accurate prognosis in primary prostate cancer.ConclusionsIn contrast to the majority of previous reports suggesting tumor suppressive functions in other cancers, our observations identify a clear oncogenic role for ING3, which acts as a co-activator of AR in prostate cancer. Data from TCGA and our previous and current tissue microarrays suggest that ING3 levels correlate with AR levels and that in patients with low levels of the receptor, ING3 level could serve as a useful prognostic biomarker.

Allele-specific non-CG DNA methylation marks domains of active chromatin in female mouse brain

DNA methylation at gene promoters in a CG context is associated with transcriptional repression, including at genes silenced on the inactive X chromosome in females. Non-CG methylation (mCH) is a distinct feature of the neuronal epigenome that is differentially distributed between males and females on the X chromosome. However, little is known about differences in mCH on the active (Xa) and inactive (Xi) X chromosomes because stochastic X-chromosome inactivation (XCI) confounds allele-specific epigenomic profiling. We used whole-genome bisulfite sequencing in a mouse model with nonrandom XCI to examine allele-specific DNA methylation in frontal cortex. Xi was largely devoid of mCH, whereas Xa contained abundant mCH similar to the male X chromosome and the autosomes. In contrast to the repressive association of DNA methylation at CG dinucleotides (mCG), mCH accumulates on Xi in domains with transcriptional activity, including the bodies of most genes that escape XCI and at the X-inactivation center, validating this epigenetic mark as a signature of transcriptional activity. Escape genes showing CH hypermethylation were the only genes with CG-hypomethylated promoters on Xi, a well-known mark of active transcription. Finally, we found extensive allele-specific mCH and mCG at autosomal imprinted regions, some with a negative correlation between methylation in the two contexts, further supporting their distinct functions. Our findings show that neuronal mCH functions independently of mCG and is a highly dynamic epigenomic correlate of allele-specific gene regulation.

Regulation of hepatic microRNA expression by hepatocyte nuclear factor 4 alpha.

AIMTo uncover the role of hepatocyte nuclear factor 4 alpha (HNF4α) in regulating hepatic expression of microRNAs.METHODSMicroarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4α expression in liver (Hnf4α-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4α, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4α on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3’UTR) of two genes in human hepatoma cells.RESULTSMicroarray data indicated that most microRNAs remained unaltered by Hnf4α deficiency in Hnf4α-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4α-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4α-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4α directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4α markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3’UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1.CONCLUSIONHNF4α is essential for hepatic basal expression of a group of liver-enriched microRNAs, including miR-101, miR-192, miR-193a, miR-194 and miR-802, through which HNF4α may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.

ING1 regulates rRNA levels by altering nucleolar chromatin structure and mTOR localization.

Epigenetic, transcriptional and signaling processes in the nucleolus regulate rRNA transcription and cell growth. We report here that the tumor suppressor ING1b binds rDNA, regulates rDNA chromatin modifications and affects nucleolar localization of mTOR to modulate rRNA levels. ING1 represses rDNA transcription by recruiting HDAC1 to rDNA loci, increasing its association with the NoRC complex and deacetylating the histone H3K9 and H3K27 marks of active transcription. Loss of ING1 enhances nucleolar localization of phospho-mTOR and its association with Raptor and GβL, even during rapamycin treatment. ING1 inhibits rDNA transcription by inhibiting UBF activity and its interaction with mTOR. Regulation of rDNA heterochromatin and rRNA synthesis by ING1 is also apparent during normal cell growth and during cell stress. Moreover, this function was also important during PMA induced differentiation of THP1 cells, since knocking down ING1 affected the process by inhibiting rRNA transcriptional repression. These observations show that ING1 regulates the nucleolar epigenome and rDNA transcription suggesting that regulation of protein synthesis might serve as the basis for ING1 function as a type II tumor suppressor.

Abstract 4533: Disruption of intrachromosomal interactions by deficient cohesin initiate epithelial-mesenchymal transition and stemness in cancer

Abstract PURPOSE Although EMT-MET process is a crucial reversible event in development of human tumorigenesis controlled by complex networks, very little is known about how EMT-MET occurs reversibly and timely in cancer. Recently, different levels of Rad21 protein, a subunit of cohesin high-order-chromatin-architectural complex, between mesenchymal and epithelial cancers have been described. METHODS To elucidate how Rad21 determines EMT states, we silenced Rad21 in epithelial cancers using lentiviral shRNAs. We performed microarray to compare transcriptional changes by Rad21-KD. Additionally, we measured cell cycles and mitotic chromosome segregations to rule out the role of Rad21 as a sister chromatid segregation molecule. We observed cell morphology through confocal microscopy and checked high-order-chromatin structures using Chromosome-Conformation-Capture (3C) and Chromatin immunoprecipitation (ChIP) assay. RESULTS We identified high-order-chromatin structures mediated by Rad21 plays critical regulatory roles in transcription of EMT related genes for the first time. Rad21-KD in epithelial cancers caused reduction of Rad21 enrichment on TGFB1 and ITGA5 genes, leading to disruption of the-gene-specific-chromatin-looping structures. This dramatically recruited PolII and AcH3, active transcription marks, causing rapid induction of the gene transcriptions. Indeed, induced EMT-related genes directly led to morphological changes with EMT traits. We ultimately found that cancer stem cell-like cells showed same results with mesenchymal cancer and Rad21-KD epithelial cells. These findings indicate that the initiation of EMT in cancer might crucially require changes in high-order-chromatin structures of EMT-related genes mediated by cohesin, contributing to rapid and precise reversible changes in EMT or MET state to adapt to phenotypes of human cancer development. Citation Format: Jiyeon Yun. Disruption of intrachromosomal interactions by deficient cohesin initiate epithelial-mesenchymal transition and stemness in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4533.

Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome.

Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes.

H3K4 tri-methylation breadth at transcription start sites impacts the transcriptome of systemic lupus erythematosus.

BackgroundThe autoimmune disease systemic lupus erythematosus (SLE) has a modified epigenome with modified tri-methylation of histone H3 lysine 4 (H3K4me3) at specific loci across the genome. H3K4me3 is a canonical chromatin mark of active transcription. Recent studies have suggested that H3K4me3 breadth has an important regulatory role in cell identity. This project examined H3K4me3 breadth at transcription start sites (TSS) in primary monocytes and its association with differential gene transcription in SLE.ResultsIntegrative analysis was applied to chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data generated from primary monocytes as well as genomic data available in public repositories. Four distinctive H3K4me3 patterns of ChIP-seq peaks were identified at 8399 TSSs. Narrow peaks were highly enriched with genes related to housekeeping functions. The broader peaks with extended H3K4me3 immediately upstream and/or downstream of TSS were associated with immune response genes. Many TSSs had downstream H3K4me3 extended to ~650 bp, where the transition of H3K4me3 to H3K36me3, a transcriptional elongation mark, is often found. The H3K4me3 pattern was strongly associated with transcription in SLE. Genes with narrow peaks were less likely (OR = 0.14, p = 2 × 10−4) while genes with extended downstream H3K4me3 were more likely (OR = 2.37, p = 1 × 10−11) to be overexpressed in SLE. Of the genes significantly overexpressed in SLE, 78.8 % had increased downstream H3K4me3 while only 47.1 % had increased upstream H3K4me3. Gene transcription sensitively and consistently responded to H3K4me3 change downstream of TSSs. Every 1 % increase of H3K4me3 in this region leads to ~1.5 % average increase of transcription.ConclusionsWe identified the immediate TSS downstream nucleosome as a crucial regulator responsible for transcription changes in SLE. This study applied a unique method to study the effect of H3K4me3 breadth on diseases and revealed new insights about epigenetic modifications in SLE, which could lead to novel treatments.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0179-4) contains supplementary material, which is available to authorized users.

Abstract 966: LSD1 functions as a global androgen receptor coactivator and is a therapeutic target in prostate cancer

Abstract Androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) and remains critical for more aggressive castration-resistant PCa (CRPC) through transactivation of multiple genes. Although CRPC is generally responsive to further CYP17A1 inhibition (abiraterone) or more potent AR antagonist (enzalutamide) treatments, patients treated with these agents still relapse within 1-2 years, and high levels of AR and AR regulated genes in many of these relapsed tumors indicate that AR activity has again been restored. Therefore, there remains a need for further novel AR targeted therapies. Initially identified as a component in the REST complex that functions as a transcriptional suppressor, Lysine Specific Demethylase 1 (LSD1) removes the methyl group from histone 3 lysine 4 (H3K4me1 and 2), and turns off the marks of active transcription. Our recent genome-wide integrated analysis of LSD1 and AR chromatin bindings and gene expression profiles indicates that LSD1 is broadly associated with AR regulated enhancers, and LSD1 functions as a coactivator on AR-stimulated genes through demethylating novel histone or nonhistone substrates in addition to H3K4me. LSD1 is also associated with FOXA1 at AR regulated enhancer sites, and an LSD1-FOXA1 interaction enhances binding of both proteins at these sites. These results clearly demonstrated LSD1 as a therapeutic target in prostate cancer. We then accessed if LSD1 enzymatic activity is required for its global AR coactivator function. AR transcription activity is significantly impaired in cells overexpressing a catalytic-deficient LSD1 mutant. In addition, this mutant also decreased FOXA1 binding on AR-regulated enhancers. We then selected of a panel of LSD1 inhibitors (a few of them have been used in clinical trials for lung cancer and leukemia) to assess the effects on AR activity and PCa cell growth. Inhibition of LSD1 consistently impaired AR activity through abrogating acetylated H3K27 at AR-regulated enhancers. LSD1 inhibitor also decreased FOXA1 binding prior to androgen stimulation. While most of inhibitors showed relatively low efficacy in PCa cells (>50μM), LSD1-C12 is highly potent and specific at μM range to dramatically decrease AR activity and PCa cell growth. Overall, our study suggests that the AR coactivator function of LSD1 depends on its enzymatic activity and targeting LSD1 with more specific and potent inhibitors is a promising therapeutic approach to treat PCa. Furthermore, understanding the mechanism of LSD1 coactivator function will potentially advance PCa therapy by targeting LSD1. Citation Format: Shuai Gao, Yanfei Gao, Hansen He, Myles Brown, Steve Balk, Changmeng Cai. LSD1 functions as a global androgen receptor coactivator and is a therapeutic target in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 966. doi:10.1158/1538-7445.AM2015-966