High-throughput sequencing technology is central to our current understanding of the human methylome. The vast majority of studies use chemical conversion to analyse bulk-level patterns of DNA ...methylation across the genome from a population of cells. While this technology has been used to probe single-molecule methylation patterns, such analyses are limited to short reads of a few hundred basepairs. DNA methylation can also be directly detected using Nanopore sequencing which can generate reads measuring megabases in length. However, thus far these analyses have largely focused on bulk-level assessment of DNA methylation. Here, we analyse DNA methylation in single Nanopore reads from human lymphoblastoid cells, to show that bulk-level metrics underestimate large-scale heterogeneity in the methylome. We use the correlation in methylation state between neighbouring sites to quantify single-molecule heterogeneity and find that heterogeneity varies significantly across the human genome, with some regions having heterogeneous methylation patterns at the single-molecule level and others possessing more homogeneous methylation patterns. By comparing the genomic distribution of the correlation to epigenomic annotations, we find that the greatest heterogeneity in single-molecule patterns is observed within heterochromatic partially methylated domains (PMDs). In contrast, reads originating from euchromatic regions and gene bodies have more ordered DNA methylation patterns. By analysing the patterns of single molecules in more detail, we show the existence of a nucleosome-scale periodicity in DNA methylation that accounts for some of the heterogeneity we uncover in long single-molecule DNA methylation patterns. We find that this periodic structure is partially masked in bulk data and correlates with DNA accessibility as measured by nanoNOMe-seq, suggesting that it could be generated by nucleosomes. Our findings demonstrate the power of single-molecule analysis of long-read data to understand the structure of the human methylome.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The aberrant gain of DNA methylation at CpG islands is frequently observed in colorectal tumours and may silence the expression of tumour suppressors such as MLH1. Current models propose that these ...CpG islands are targeted by de novo DNA methyltransferases in a sequence-specific manner, but this has not been tested. Using ectopically integrated CpG islands, here we find that aberrantly methylated CpG islands are subject to low levels of de novo DNA methylation activity in colorectal cancer cells. By delineating DNA methyltransferase targets, we find that instead de novo DNA methylation activity is targeted primarily to CpG islands marked by the histone modification H3K36me3, a mark associated with transcriptional elongation. These H3K36me3 marked CpG islands are heavily methylated in colorectal tumours and the normal colon suggesting that de novo DNA methyltransferase activity at CpG islands in colorectal cancer is focused on similar targets to normal tissues and not greatly remodelled by tumourigenesis.
DNA repair defects underlie many cancer syndromes. We tested whether de novo germline mutations (DNMs) are increased in families with germline defects in polymerase proofreading or base excision ...repair. A parent with a single germline POLE or POLD1 mutation, or biallelic MUTYH mutations, had 3-4 fold increased DNMs over sex-matched controls. POLE had the largest effect. The DNMs carried mutational signatures of the appropriate DNA repair deficiency. No DNM increase occurred in offspring of MUTYH heterozygous parents. Parental DNA repair defects caused about 20-150 DNMs per child, additional to the ~60 found in controls, but almost all extra DNMs occurred in non-coding regions. No increase in post-zygotic mutations was detected, excepting a child with bi-allelic MUTYH mutations who was excluded from the main analysis; she had received chemotherapy and may have undergone oligoclonal haematopoiesis. Inherited DNA repair defects associated with base pair-level mutations increase DNMs, but phenotypic consequences appear unlikely.
The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved by the de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3B localises to H3K36me3 at ...actively transcribing gene bodies via its PWWP domain. It also functions at heterochromatin through an unknown recruitment mechanism. Here, we find that knockout of DNMT3B causes loss of methylation predominantly at H3K9me3-marked heterochromatin and that DNMT3B PWWP domain mutations or deletion result in striking increases of methylation in H3K9me3-marked heterochromatin. Removal of the N-terminal region of DNMT3B affects its ability to methylate H3K9me3-marked regions. This region of DNMT3B directly interacts with HP1α and facilitates the bridging of DNMT3B with H3K9me3-marked nucleosomes in vitro. Our results suggest that DNMT3B is recruited to H3K9me3-marked heterochromatin in a PWWP-independent manner that is facilitated by the protein’s N-terminal region through an interaction with a key heterochromatin protein. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in cancer, aging and Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome.
Synopsis
The DNA methyltransferase DNMT3B is recruited to gene bodies through its PWWP domain’s interaction with H3K36me3. This study shows DNMT3B PWWP mutations cause hypermethylation of H3K9me3-marked heterochromatin facilitated by DNMT3B’s N-terminal region.
Removal of DNMT3B results in losses of DNA methylation from H3K9me3-methylated heterochromatin.
Mutation or deletion of DNMT3B’s PWWP domain results in the hypermethylation of H3K9me3-marked heterochromatin.
Recruitment of DNMT3B to H3K9me3 is facilitated by its N-terminal region, which interacts with HP1α.
The DNA methyltransferase DNMT3B is recruited to gene bodies through its PWWP domain’s interaction with H3K36me3. This study shows DNMT3B PWWP mutations cause hypermethylation of H3K9me3-marked heterochromatin facilitated by DNMT3B’s N-terminal region.
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•The hippocampus-prefrontal cortex circuit plays a major role in stress and depression.•Nucleus Reuniens lesion prevents the hippocampal neuronal atrophy induced by chronic mild ...stress.•The antidepressant sertraline reverses the hippocampal neuronal atrophy induced by chronic mild stress.
The hippocampus-prefrontal cortex circuit plays a major role in stress and in the neurobiology of depression and its treatment. Disruption of this circuit by lesioning the thalamic nucleus reuniens (RE) has been shown to prevent the detrimental effects of chronic mild stress on prefrontal cortex neuroplasticity indices in male rats. However, it remains unknown whether hippocampal neurostructural response to stress is modified by RE lesion. In the present study, adult male rats were subjected to the chronic mild stress model of depression and were treated with either vehicle or an antidepressant (i.e. sertraline). Moreover, a group of animals was subjected to RE lesion before stress exposure with or without sertraline treatment. We demonstrated that chronic mild stress induced hippocampal CA1 dendritic atrophy and this was prevented by pre-stress RE lesion to the same extent that antidepressant treatment reversed it. The present findings highlight the importance of hippocampal-prefrontal cortex communication in chronic stress effects on hippocampal neuroplasticity and contribute to the elucidation of the role of RE in neurostructural changes underlying stress-driven depression and its treatment.
Olive (Olea europaea L.) cultivation is rapidly expanding and low quality saline water is often used for irrigation. The molecular basis of salt tolerance in olive, though, has not yet been ...investigated at a system level. In this study a comparative transcriptomics approach was used as a tool to unravel gene regulatory networks underlying salinity response in olive trees by simulating as much as possible olive growing conditions in the field. Specifically, we investigated the genotype-dependent differences in the transcriptome response of two olive cultivars, a salt-tolerant and a salt-sensitive one.
A 135-day long salinity experiment was conducted using one-year old trees exposed to NaCl stress for 90 days followed by 45 days of post-stress period during the summer. A cDNA library made of olive seedling mRNAs was sequenced and an olive microarray was constructed. Total RNA was extracted from root samples after 15, 45 and 90 days of NaCl-treatment as well as after 15 and 45 days of post-treatment period and used for microarray hybridizations. SAM analysis between the NaCl-stress and the post-stress time course resulted in the identification of 209 and 36 differentially expressed transcripts in the salt-tolerant and salt-sensitive cultivar, respectively. Hierarchical clustering revealed two major, distinct clusters for each cultivar. Despite the limited number of probe sets, transcriptional regulatory networks were constructed for both cultivars while several hierarchically-clustered interacting transcription factor regulators such as JERF and bZIP homologues were identified.
A systems biology approach was used and differentially expressed transcripts as well as regulatory interactions were identified. The comparison of the interactions among transcription factors in olive with those reported for Arabidopsis might indicate similarities in the response of a tree species with Arabidopsis at the transcriptional level under salinity stress.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•Both whole exome sequencing and genotyping was performed in the Cretan Aging Cohort.•p.Ser498Ala GLUD2 variant was more common in controls than in AD patients (p < 0.01).•The TARDBP p.Ile383Val ...variant was found in 2 patients diagnosed with AD.•TARDBP p.Ile383Val was found in 1 patient with ALS and 1 patient with ALS/FTD.
Using exome sequencing, we analyzed 196 participants of the Cretan Aging Cohort (CAC; 95 with Alzheimer's disease AD, 20 with mild cognitive impairment MCI, and 81 cognitively normal controls). The APOE ε4 allele was more common in AD patients (23.2%) than in controls (7.4%; p < 0.01) and the PSEN2 p.Arg29His and p.Cys391Arg variants were found in 3 AD and 1 MCI patient, respectively. Also, we found the frontotemporal dementia (FTD)-associated TARDBP gene p.Ile383Val variant in 2 elderly patients diagnosed with AD and in 2 patients, non CAC members, with the amyotrophic lateral sclerosis/FTD phenotype. Furthermore, the p.Ser498Ala variant in the positively selected GLUD2 gene was less frequent in AD patients (2.11%) than in controls (16%; p < 0.01), suggesting a possible protective effect. While the same trend was found in another local replication cohort (n = 406) and in section of the ADNI cohort (n = 808), this finding did not reach statistical significance and therefore it should be considered preliminary. Our results attest to the value of genetic testing to study aged adults with AD phenotype.
Abstract
Aim: Studying exceptional responders to standard treatments may provide broader insights into the genetic mechanisms underlying such responses and suggest therapeutic options that weren’t ...previously apparent. In this study we unravelled the genetic signature of undifferentiated sarcoma in order to disclose the molecular mechanisms that may underlie the patient’s exquisite sensitivity to Pazopanib.
Methods: A female patient with massive undifferentiated gynecological sarcoma experienced a dramatic response to Pazopanib that made debulking surgery possible. We performed whole exome sequencing on DNA isolated from both blood and formalin fixed paraffin-embedded (FFPE) tumour employing Ion Torrent technology. Variant annotation was performed using the Ion ReporterTM v5.0 (MA, USA) tool after application of specific filters. Subsequent curation of annotated data was completed using a combined semi-automated data analyses workflow developed by Minotech Genomics Facility with the implementation of two in house-developed lists regarding 24 pazopanib-related genes, and 96 angiogenesis-associated genes. We also performed targeted sequencing of Ion Ampliseq Comprehensive Cancer panel (CCP) that is strategically designed to interrogate coding DNA sequences and splice variants across 409 tumour suppressor genes and oncogenes.
Results: Whole exome sequencing was conducted at 20x coverage for 91.9% and 60.5% of >19,000 genes for germline and FFPE DNA samples, respectively. Targeted sequencing was conducted at 20x for >97% of 409 genes of the comprehensive cancer panel. Comparative analyses revealed two variants common in germline and FFPE samples, localized in LTK(p.Val541Ile) and FGFR4 (p.Gly388Arg). Interestingly, examination of the annotated data for biologically plausible mechanisms revealed 2 yet uncharacterized heterozygous variants in two other members of the FGFR family, FGFR2 (c.2183A>G, p.Asn728Ser) and FGFR3 (c.2050G>A, p.Val684Ile) that were present only in the FFPE samples. Even though both variants reside within the catalytic tyrosine kinase domains, they are predicted to exert tolerated effects on the encoded FGFR2 and FGFR3 proteins. Since, Asn728Ser is highly conserved among different kinases of the FGFR family we chose this variant to predict a possible effect on Pazopanib binding. Specifically, protein structure simulation of FGFR2 revealed that asparagine residue at position 728 is located in the external side of the drug binding cavity and is thus predicted to not affect Pazopanib binding.
Conclusion: We pursue the possible genetic or somatic modifiers underling the excessive response to Pazopanib. The detection of two, yet uncharacterised, variants in known Pazopanib targets highlights the potential of next generation sequencing in disclosing the genetic signature of undifferentiated sarcomas in relation to the activity and effectiveness of certain therapeutic agents.
Note: This abstract was not presented at the meeting.
Citation Format: Emmanouil Saloustros, Helen Latsoudis, Despoina Vassou, Galateia Kallergi, Irene Stratidaki, Niki-Antonia Gounalaki, Vasiliki Fadouloglou, Ioannis Drositis, Emmanouil Kontopodis, Kitty Pavlakis, Dimitrios Mavroudis, Nikolaos Androulakis, Dimitrios Kafetzopoulos. The pursue of genetic mechanisms underlying supreme response to pazopanib treatment abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3740. doi:10.1158/1538-7445.AM2017-3740
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