Podcasts about h3k4me3

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Best podcasts about h3k4me3

Latest podcast episodes about h3k4me3

Oncotarget
Differential Expression of Mad2 Gene in Human Esophageal Cancer

Oncotarget

Play Episode Listen Later Feb 12, 2024 3:02


BUFFALO, NY- February 12, 2024 – A new research paper was published in Oncotarget's Volume 15 on February 5, 2024, entitled, “Differential expression of Mad2 gene is consequential to the patterns of histone H3 post-translational modifications in its promoter region in human esophageal cancer samples.” Raw areca nut (AN) consumption increases esophageal squamous cell carcinoma (ESCC) due to overexpression of securin (pituitary tumor transforming gene1), causing chromosomal instability. Mitotic arrest deficient protein 2 (Mad2), a crucial spindle assembly checkpoint protein, is at risk of aneuploidy and tumor development when overexpressed or underexpressed. In this new study, researchers Chongtham Sovachandra Singh, Nabamita Boruah, Atanu Banerjee, Sillarine Kurkalang, Pooja Swargiary, Hughbert Dakhar, and Anupam Chatterjee from The Assam Royal Global University, University of Pennsylvania, LN Mithila University, University of Chicago Medicine, Nazareth Hospital, Laitumkhrah, and North-Eastern Hill University evaluated Mad2 status in human ESCC with AN consumption habits, revealing unclear molecular mechanisms. Human ESCC samples (n = 99) were used for loss of heterozygosity analysis at 4q25-28, while 32 samples were used for expression analysis of Mad2, E2F1 genes, and Rb-phosphorylation. Blood samples were used for metaphase preparation. The Mad2 deregulation was assessed using chromatin immunoprecipitation-qPCR assay in the core promoter region, establishing its association with the pRb-E2F1 circuit for the first time. “The study revealed overexpression and underexpression of Mad2, premature anaphase, and chromosome missegregation in all the samples.” LOH pattern identified a deletion in D4S2975 in 40% of ESCC samples. The study reveals the deregulation of pRb-E2F1 circuit in all samples. 4q27 disruption could be a factor for Mad2 underexpression in AN-induced esophageal carcinogenesis, while overexpression may be due to the deregulation of the Rb-E2F1 circuit and consequently elevation of H3K4me3 and H3K9ac. “Mad2 expression levels with chromosomal abnormalities can be a clinical biomarker, but further research is needed to understand pRb's role in Mad2 down-regulation.” DOI - https://doi.org/10.18632/oncotarget.28554 Correspondence to - Anupam Chatterjee - achatterjee@rgu.ac, chatterjeeanupam@hotmail.com Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28554 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, Mad2 gene, histone methylation, histone acetylation, Rb-phosphorylation; esophageal cancer About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Spotify - https://open.spotify.com/show/0gRwT6BqYWJzxzmjPJwtVh Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Authentic Biochemistry
ImmunoEpigenetics96. APC-agMHCII and Bcell:Tcell synapse interactions yield subsequent plasma cell antibody production under the H3K4me3 epigenetic alteration of chromatin accessibility.DJGPhD.23.7.23

Authentic Biochemistry

Play Episode Listen Later Jul 23, 2023 29:18


References J Immunol. 2014 Aug 15; 193(4): 1531–1537 Immunology lecture notes-Guerra --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message

PaperPlayer biorxiv cell biology
The BOD1L subunit of the mammalian SETD1A complex sustains the expression of DNA damage repair genes despite restraining H3K4 trimethylation

PaperPlayer biorxiv cell biology

Play Episode Listen Later Apr 6, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535882v1?rss=1 Authors: Ciotta, G., Singh, S., Gupta, A., Torres, D. C., Fu, J., Choudhury, R., Chu, W. K., Choudhary, C., Gahurova, L., Al-Fatlawi, A., Schroeder, M., Aasland, R., Poetsch, A., Anastassiadis, K., Stewart, A. F. Abstract: SETD1A is the histone 3 lysine 4 (H3K4) methyltransferase central to the mammalian version of the highly conserved eight subunit Set1 complex (Set1C) that apparently conveys H3K4 trimethylation (H3K4me3) onto all active Pol II promoters. Accordingly, mouse embryonic stem cells (ESCs) die when SETD1A is removed. We report that death is accompanied by loss of expression of DNA repair genes and accumulating DNA damage. BOD1L and BOD1 are homologs of the yeast Set1C subunit, Shg1, and subunits of the mammalian SETD1A and B complexes. We show that the Shg1 homology region binds to a highly conserved central a-helix in SETD1A and B. Like mutagenesis of Shg1 in yeast, conditional mutagenesis of Bod1l in ESCs promoted increased H3K4 di- and tri-methylation but also, like loss of SETD1A, loss of expression of DNA repair genes, increased DNA damage and cell death. In contrast to similar losses of gene expression, the converse changes in H3K4 methylation implies that H3K4 methylation is not essential for expression of the DNA repair network genes. Because BOD1L becomes highly phosphorylated after DNA damage and acts to protect damaged replication forks, the SETD1A complex and BOD1L in particular are key nodes for the DNA damage repair network. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Epigenetics Podcast
The Epigenetics of Human Sperm Cells (Sarah Kimmins)

Epigenetics Podcast

Play Episode Listen Later Mar 9, 2023 44:41


In this episode of the Epigenetics Podcast, we caught up with Sarah Kimmins from Université de Montreal to talk about her work on the epigenetics of human sperm cells. The focus of Sarah Kimmins and her lab is how sperm and offspring health is impacted by the father's environment. The core of this is the sperm epigenome, which has been implicated in complex diseases such as infertility, cancer, diabetes, schizophrenia and autism. The Kimmins lab is interested which players play a role in this and came across the Histone post-translational modification H3K4me3. In this interview we talk about how the father's life choices can impact offspring health, which can also be inherited transgenerationally and how this can be used to develop intervention strategies to improve child and adult health.   References Siklenka, K., Erkek, S., Godmann, M., Lambrot, R., McGraw, S., Lafleur, C., Cohen, T., Xia, J., Suderman, M., Hallett, M., Trasler, J., Peters, A. H., & Kimmins, S. (2015). Disruption of histone methylation in developing sperm impairs offspring health transgenerationally. Science (New York, N.Y.), 350(6261), aab2006. https://doi.org/10.1126/science.aab2006 Lismer, A., Siklenka, K., Lafleur, C., Dumeaux, V., & Kimmins, S. (2020). Sperm histone H3 lysine 4 trimethylation is altered in a genetic mouse model of transgenerational epigenetic inheritance. Nucleic acids research, 48(20), 11380–11393. https://doi.org/10.1093/nar/gkaa712 Lismer, A., Dumeaux, V., Lafleur, C., Lambrot, R., Brind'Amour, J., Lorincz, M. C., & Kimmins, S. (2021). Histone H3 lysine 4 trimethylation in sperm is transmitted to the embryo and associated with diet-induced phenotypes in the offspring. Developmental cell, 56(5), 671–686.e6. https://doi.org/10.1016/j.devcel.2021.01.014   Related Episodes H3K4me3, SET Proteins, Isw1, and their Role in Transcription (Jane Mellor) The Effects of Early Life Stress on Mammalian Development (Catherine J. Peña) DNA Methylation and Mammalian Development (Déborah Bourc'his)   Contact Epigenetics Podcast on Twitter Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Active Motif on Twitter Active Motif on LinkedIn Email: podcast@activemotif.com

PaperPlayer biorxiv cell biology
Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation

PaperPlayer biorxiv cell biology

Play Episode Listen Later Mar 1, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.28.530230v1?rss=1 Authors: Harris, R. J., Heer, M., Levasseur, M. D., Cartwright, T. N., Weston, B., Mitchell, J. L., Coxhead, J. M., Gaughan, L., Prendergast, L., Rico, D., Higgins, J. M. G. Abstract: Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where particular combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognise trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me3 during mitosis. We find that H3K4me3 hinders adjacent H3T3ph deposition in cells, and that the PHD domain of TAF3 can bind H3K4me3 in mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

PaperPlayer biorxiv neuroscience
Histone bivalency regulates the timing of cerebellar granule cell development

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Feb 3, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.02.526881v1?rss=1 Authors: Mätlik, K., Govek, E.-E., Paul, M. R., Allis, C. D., Hatten, M. E. Abstract: Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature, multipolar neurons. Here we use RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron, the mouse cerebellar granule cell (GC). We find that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent H3K4me3 and H3K27me3 domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes and induced the downregulation of migration-related genes and upregulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Epigenetics Podcast
The Role of PHF13 in Chromatin and Transcription (Sarah Kinkley)

Epigenetics Podcast

Play Episode Listen Later Jan 12, 2023 34:33


In this episode of the Epigenetics Podcast, we caught up with Sarah Kinkley from the Max Planck Institute of Molecular Genetics to talk about her work on PHF13 and its role in chromatin and transcription. The Kinkley laboratory focuses mainly on unraveling the mechanism of action of the transcription factor PHF13 (PHC Finger Protein 13). PHF13 is a reader of the epigenetic mark H3K4 trimethylation which influences higher chromatin order, transcriptional regulation, and differentiation. The lab has shown that PHF13 plays a crucial role in phase separation and mitotic chromatin compaction.   References Kinkley, S., Staege, H., Mohrmann, G., Rohaly, G., Schaub, T., Kremmer, E., Winterpacht, A., & Will, H. (2009). SPOC1: a novel PHD-containing protein modulating chromatin structure and mitotic chromosome condensation. Journal of cell science, 122(Pt 16), 2946–2956. https://doi.org/10.1242/jcs.047365 Chung, H. R., Xu, C., Fuchs, A., Mund, A., Lange, M., Staege, H., Schubert, T., Bian, C., Dunkel, I., Eberharter, A., Regnard, C., Klinker, H., Meierhofer, D., Cozzuto, L., Winterpacht, A., Di Croce, L., Min, J., Will, H., & Kinkley, S. (2016). PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3. eLife, 5, e10607. https://doi.org/10.7554/eLife.10607 Connecting the Dots: PHF13 and cohesin promote polymer-polymer phase separation of chromatin into chromosomes. Francesca Rossi, Rene Buschow, Laura V. Glaser, Tobias Schubert, Hannah Staege, Astrid Grimme, Hans Will, Thorsten Milke, Martin Vingron, Andrea M. Chiariello, Sarah Kinkley. bioRxiv 2022.03.04.482956; doi: https://doi.org/10.1101/2022.03.04.482956   Related Episodes The Role of Blimp-1 in Immune-Cell Differentiation (Erna Magnúsdóttir) H3K4me3, SET Proteins, Isw1, and their Role in Transcription (Jane Mellor) The Role of SMCHD1 in Development and Disease (Marnie Blewitt)   Contact Epigenetics Podcast on Twitter Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Active Motif on Twitter Active Motif on LinkedIn Email: podcast@activemotif.com

PaperPlayer biorxiv neuroscience
Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates the axon guidance genes robo2 and slit in Drosophila

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Jan 7, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.03.522592v1?rss=1 Authors: Singh, B. N., Tran, H., Kramer, J., Kirishenko, E., Changela, N., Wang, F., Feng, Y., Kumar, D., Tu, M., Liang, S., Lan, J., Bizet, M., Fuks, F., Steward, R. Abstract: Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases enzymes catalyzing the transition of 5mC to 5hmC in DNA and have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila because Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by determining Tet DNA-binding sites throughout the genome and by mapping the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC-modified sites can be found along the entire transcript and are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are frequently involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and are sensitized to reduced levels of slit. Both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs, primarily in developing nerve cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

PaperPlayer biorxiv cell biology
MYSM1 co-activates ERα action via histone and non-histone deubiquitination to confer antiestrogen resistance in breast cancer

PaperPlayer biorxiv cell biology

Play Episode Listen Later Dec 24, 2022


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.23.521780v1?rss=1 Authors: Zhao, Y., Luan, R., Sun, G., Zhou, B., Wang, M., Bai, Y., Wang, C., Wang, S., Zeng, K., Feng, J., He, M., Lin, L., Wei, Y., Zhang, Q. Abstract: Endocrine resistance is a crucial challenge in estrogen receptor alpha (ER)-positive breast cancer (BCa) therapy. Aberrant alteration in modulation of E2/ER signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Thus, identification the efficient ER cofactor remains necessary for finding a potential therapeutic target for endocrine resistance. Herein, we have demonstrated that Myb like, SWIRM and MPN domains 1 (MYSM1) as a histone deubiquitinase is a novel ER co-activator with established Drosophila experimental model. Our results showed that MYSM1 participated in up-regulation of ER action via histone and non-histone deubiquitination. We provided the evidence to show that MYSM1 was involved in maintenance of ER stability via ER deubiquitination. Furthermore, silencing MYSM1 induced enhancement of histone H2A ubiquitination as well as reduction of histone H3K4me3 and H3Ac levels at cis regulatory elements on promoter of ER-regulated gene. In addition, MYSM1 depletion attenuated cell proliferation/growth in BCa-derived cell lines and xenograft models. Knockdown of MYSM1 increased the sensitivity of antiestrogen agents in BCa cells. MYSM1 was highly expressed in clinical BCa samples, especially in aromatase inhibitor (AI) non-responsive tissues. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ER action and provide a potential therapeutic target for endocrine resistance in BCa. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

The Nonlinear Library
LW - What is epigenetics? by Metacelsus

The Nonlinear Library

Play Episode Listen Later Nov 6, 2022 12:04


Welcome to The Nonlinear Library, where we use Text-to-Speech software to convert the best writing from the Rationalist and EA communities into audio. This is: What is epigenetics?, published by Metacelsus on November 6, 2022 on LessWrong. Among all areas of biology related to my research, epigenetics is the one that is most commonly misunderstood, not only by the general public but even by other scientists. After being irritated one too many times, I've decided to make a series of posts to explain what epigenetics really is, why it's important, and how it's misunderstood. I will also explain how epigenetics is important for my own research on making gametes from stem cells. This first post covers the definition of epigenetics, and the basic biology of epigenetic marks. What is genetics? Before defining epigenetics, let's start with a definition of genetics. Genetics is the study of genes, which are sequences of genetic material that encode functional products. Let's take the IGF2 gene as an example. Depicted above is a region of human chromosome 11 containing the IGF2 gene, which encodes the IGF2 protein, an important growth factor for fetal development. The boxes represent exons and lines represent introns. The darker green color is the protein-coding sequence, and non-coding (i.e. untranslated) regions are shown in lighter green. Arrows represent the direction of transcription. The bottom of this image shows the location of common genetic variants (present at >1% frequency). If you look closely, you might notice that none of them are in the protein-coding sequence (the dark green boxes). This is not a coincidence, because nothing is ever a coincidence most mutations to essential proteins (including IGF2) are harmful and thus selected out of the population. However, there are several common mutations in non-coding regions of this gene. To recap, genetics is the study of genes (such as IGF2) and the effects of genetic variation on their functions. What is epigenetics? Epigenetics is the study of epigenetic marks, which are changes to genetic material that alter gene expression, but do not change the genetic sequence. A decent analogy for epigenetic marks is CAPITALIZATION, bolding, or strikethroughs in text. DNA methylation and histone modifications are the two kinds of epigenetic marks. Some people also consider long noncoding RNAs (such as those involved in X-chromosome inactivation) to be epigenetic marks. Although these RNAs are undoubtedly important for regulating gene expression, I would not classify them as epigenetic marks since they are not direct modifications to genetic material. In vertebrate animals, the cytosine in CG sequences often has a methyl group attached, forming 5-methylcytosine. A CG sequence is also CG on the opposite strand, so the cytosines on both strands can be methylated. To make things confusing, methylation at CG sequences is termed CpG methylation, the lowercase p standing for phosphate. 5-methylcytosine will pair with guanine just like normal cytosine, but it is not equivalent to cytosine in its interactions with DNA-binding proteins. Generally, CpG methylation suppresses the expression of nearby genes. CpG sites often cluster together to form “CpG islands” in important regulatory regions. Other organisms (invertebrates, plants, fungi, bacteria) have different ways of methylating DNA. I won't get into them in this post series, but you should know that CpG methylation is not universal. Modifications to histones are another important set of epigenetic marks. Histones are DNA packaging proteins, which form complexes called nucleosomes. DNA winds around nucleosomes sort of like thread around spools. The overall assembly of DNA and histones is known as chromatin. Chemical modifications to histones are important epigenetic marks that can have drastic changes on gene expression. For example, trimethylation of lysine 4 on histone H3 (known as H3K4me3) marks promoters of actively transcribed genes. ...

The Nonlinear Library: LessWrong
LW - What is epigenetics? by Metacelsus

The Nonlinear Library: LessWrong

Play Episode Listen Later Nov 6, 2022 12:04


Link to original articleWelcome to The Nonlinear Library, where we use Text-to-Speech software to convert the best writing from the Rationalist and EA communities into audio. This is: What is epigenetics?, published by Metacelsus on November 6, 2022 on LessWrong. Among all areas of biology related to my research, epigenetics is the one that is most commonly misunderstood, not only by the general public but even by other scientists. After being irritated one too many times, I've decided to make a series of posts to explain what epigenetics really is, why it's important, and how it's misunderstood. I will also explain how epigenetics is important for my own research on making gametes from stem cells. This first post covers the definition of epigenetics, and the basic biology of epigenetic marks. What is genetics? Before defining epigenetics, let's start with a definition of genetics. Genetics is the study of genes, which are sequences of genetic material that encode functional products. Let's take the IGF2 gene as an example. Depicted above is a region of human chromosome 11 containing the IGF2 gene, which encodes the IGF2 protein, an important growth factor for fetal development. The boxes represent exons and lines represent introns. The darker green color is the protein-coding sequence, and non-coding (i.e. untranslated) regions are shown in lighter green. Arrows represent the direction of transcription. The bottom of this image shows the location of common genetic variants (present at >1% frequency). If you look closely, you might notice that none of them are in the protein-coding sequence (the dark green boxes). This is not a coincidence, because nothing is ever a coincidence most mutations to essential proteins (including IGF2) are harmful and thus selected out of the population. However, there are several common mutations in non-coding regions of this gene. To recap, genetics is the study of genes (such as IGF2) and the effects of genetic variation on their functions. What is epigenetics? Epigenetics is the study of epigenetic marks, which are changes to genetic material that alter gene expression, but do not change the genetic sequence. A decent analogy for epigenetic marks is CAPITALIZATION, bolding, or strikethroughs in text. DNA methylation and histone modifications are the two kinds of epigenetic marks. Some people also consider long noncoding RNAs (such as those involved in X-chromosome inactivation) to be epigenetic marks. Although these RNAs are undoubtedly important for regulating gene expression, I would not classify them as epigenetic marks since they are not direct modifications to genetic material. In vertebrate animals, the cytosine in CG sequences often has a methyl group attached, forming 5-methylcytosine. A CG sequence is also CG on the opposite strand, so the cytosines on both strands can be methylated. To make things confusing, methylation at CG sequences is termed CpG methylation, the lowercase p standing for phosphate. 5-methylcytosine will pair with guanine just like normal cytosine, but it is not equivalent to cytosine in its interactions with DNA-binding proteins. Generally, CpG methylation suppresses the expression of nearby genes. CpG sites often cluster together to form “CpG islands” in important regulatory regions. Other organisms (invertebrates, plants, fungi, bacteria) have different ways of methylating DNA. I won't get into them in this post series, but you should know that CpG methylation is not universal. Modifications to histones are another important set of epigenetic marks. Histones are DNA packaging proteins, which form complexes called nucleosomes. DNA winds around nucleosomes sort of like thread around spools. The overall assembly of DNA and histones is known as chromatin. Chemical modifications to histones are important epigenetic marks that can have drastic changes on gene expression. For example, trimethylation of lysine 4 on histone H3 (known as H3K4me3) marks promoters of actively transcribed genes. ...

PaperPlayer biorxiv neuroscience
MYT1L is required for suppressing earlier neuronal development programs in the adult mouse brain

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Oct 18, 2022


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512591v1?rss=1 Authors: Chen, J., Fuhler, N. A., Noguchi, K., Dougherty, J. D. Abstract: In vitro studies indicate the neurodevelopmental disorder gene Myelin Transcription Factor 1 Like (MYT1L) suppresses non-neuronal lineage genes during fibroblast-to-neuron direct differentiation. However, MYT1L's molecular and cellular functions during differentiation in the mammalian brain have not been fully characterized. Here, we found that MYT1L loss leads to up-regulated deep layer (DL) but down-regulated upper layer (UL) neuron gene expression, corresponding to an increased ratio of DL/UL neurons in mouse cortex. To define potential mechanisms, we conducted Cleavage Under Targets & Release Using Nuclease (CUT&RUN) to map MYT1L binding targets in mouse developing cortex and adult prefrontal cortex (PFC), and to map epigenetic changes due to MYT1L mutation. We found MYT1L mainly binds to open chromatin, but with different transcription factor co-occupancies between promoters and enhancers. Likewise, multi-omic dataset integration revealed that, at promoters, MYT1L loss does not change chromatin accessibility but does increase H3K4me3 and H3K27ac, activating both a subset of earlier neuronal development genes as well as Bcl11b, a key regulator for DL neuron development. Meanwhile, we discovered that MYT1L normally represses the activity of neurogenic enhancers associated with neuronal migration and neuronal projection development by closing chromatin structures and promoting removal of active histone marks. Further, we show MYT1L interacts with SIN3B and HDAC2 in vivo, providing potential mechanisms underlying any repressive effects on histone acetylation and gene expression. Overall, our findings provide a comprehensive map of MYT1L binding in vivo and mechanistic insights to how MYT1L facilitates neuronal maturation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Aging-US
Time Makes Histone H3 Modifications Drift In Mouse Liver

Aging-US

Play Episode Listen Later Jul 1, 2022 3:44


A new research paper was published in Aging (Aging-US) on the cover of Volume 14, Issue 12, entitled, “Time makes histone H3 modifications drift in mouse liver.” Aging is known to involve epigenetic histone modifications, which are associated with transcriptional changes, occurring throughout the entire lifespan of an individual. “So far, no study discloses any drift of histone marks in mammals which is time-dependent or influenced by pro-longevity caloric restriction treatment.” To detect the epigenetic drift of time passing, researchers—from Istituto di Ricovero e Cura a Carattere Scientifico, University of Urbino ‘Carlo Bo', University of Milan, and University of Padua—determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. “In this study, we used chromatin immunoprecipitation sequencing technology to acquire 108 high-resolution profiles of H3K4me3, H3K4me1, H3K27me3 and H3K27ac from the livers of mice aged between 3 months and 12 months and fed 30% caloric restriction diet (CR) or standard diet (SD).” The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions. “In conclusion, while our data do not establish that the observed changes in H3 modification are causally involved in aging, they indicate age, buffered by caloric restriction, releases the histone H3 marking process of transcriptional suppression in gene desert regions of mouse liver genome most of which remain to be functionally understood.” DOI: https://doi.org/10.18632/aging.204107 Corresponding Author: Marco Giorgio - marco.giorgio@unipd.it Keywords: epigenetics, aging, histones, ChIP-seq, diet Sign up for free Altmetric alerts about this article: https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.204107 About Aging-US: Launched in 2009, Aging (Aging-US) publishes papers of general interest and biological significance in all fields of aging research and age-related diseases, including cancer—and now, with a special focus on COVID-19 vulnerability as an age-dependent syndrome. Topics in Aging go beyond traditional gerontology, including, but not limited to, cellular and molecular biology, human age-related diseases, pathology in model organisms, signal transduction pathways (e.g., p53, sirtuins, and PI-3K/AKT/mTOR, among others), and approaches to modulating these signaling pathways. Follow Aging on social media: SoundCloud – https://soundcloud.com/Aging-Us Facebook – https://www.facebook.com/AgingUS/ Twitter – https://twitter.com/AgingJrnl Instagram – https://www.instagram.com/agingjrnl/ YouTube – https://www.youtube.com/agingus​ LinkedIn – https://www.linkedin.com/company/aging/ Pinterest – https://www.pinterest.com/AgingUS/ For media inquiries, please contact media@impactjournals.com

Epigenetics Podcast
The Role of Histone Dopaminylation and Serotinylation in Neuronal Plasticity (Ian Maze)

Epigenetics Podcast

Play Episode Listen Later May 12, 2022 33:53


In this episode of the Epigenetics Podcast, we caught up with Ian Maze from Ichan School of Medicine at Mount Sinai and a Howard Hughes Medical Institute (HHMI) Investigator to talk about his work on the role of histone dopaminylation and serotinylation in neuronal plasticity. The Maze group focuses on understanding the complex interplay between chromatin regulatory mechanisms in brain and neuronal plasticity. The lab places an emphasis on psychiatric disorders associated with monoaminergic (e.g., serotonin, dopamine, etc.) dysfunction, such as major depressive disorder and drug addiction. In particular the Maze team has investigated cocaine addiction and its effect on chromatin by serotonylation and dopaminylation of Histone H3 Tails.   References Maze, I., Covington, H. E., Dietz, D. M., LaPlant, Q., Renthal, W., Russo, S. J., Mechanic, M., Mouzon, E., Neve, R. L., Haggarty, S. J., Ren, Y., Sampath, S. C., Hurd, Y. L., Greengard, P., Tarakhovsky, A., Schaefer, A., & Nestler, E. J. (2010). Essential Role of the Histone Methyltransferase G9a in Cocaine-Induced Plasticity. Science, 327(5962), 213–216. https://doi.org/10.1126/science.1179438 Farrelly, L. A., Thompson, R. E., Zhao, S., Lepack, A. E., Lyu, Y., Bhanu, N. V., Zhang, B., Loh, Y.-H. E., Ramakrishnan, A., Vadodaria, K. C., Heard, K. J., Erikson, G., Nakadai, T., Bastle, R. M., Lukasak, B. J., Zebroski, H., Alenina, N., Bader, M., Berton, O., … Maze, I. (2019). Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3. Nature, 567(7749), 535–539. https://doi.org/10.1038/s41586-019-1024-7 Lepack, A. E., Werner, C. T., Stewart, A. F., Fulton, S. L., Zhong, P., Farrelly, L. A., Smith, A. C. W., Ramakrishnan, A., Lyu, Y., Bastle, R. M., Martin, J. A., Mitra, S., O'Connor, R. M., Wang, Z.-J., Molina, H., Turecki, G., Shen, L., Yan, Z., Calipari, E. S., … Maze, I. (2020). Dopaminylation of histone H3 in ventral tegmental area regulates cocaine seeking. Science, 368(6487), 197–201. https://doi.org/10.1126/science.aaw8806   Related Episodes Development of Integrative Machine Learning Tools for Neurodegenerative Diseases (Enrico Glaab) Epigenetic Influence on Memory Formation and Inheritance (Isabelle Mansuy) CpG Islands, DNA Methylation, and Disease (Sir Adrian Bird)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

Epigenetics Podcast
H3K4me3, Set-Proteins, Isw1 and their Role in Transcription (Jane Mellor)

Epigenetics Podcast

Play Episode Listen Later Jan 13, 2022 42:12


In this episode of the Epigenetics Podcast, we caught up with Jane Mellor from the University of Oxford to talk about her work on H3K4me3, SET proteins, Isw1 and their role in transcription. Since the beginning of the century, Jane Mellor and her team have focused on H3K4 trimethylation and the factors that influence this mark. They discovered that H3K4me3 is an almost universal mark of the first nucleosome in every transcribed unit and all organisms. She could subsequently, together with the Kouzarides lab, identify SetD1, the enzyme that is responsible for writing this modification. Later on, the team characterized Isw1, a chromatin remodeler which “reads” H3K4me3. More recently the lab focuses on how the polymerase transcribes throughout the first nucleosomes of the transcribed region at the +2 nucleosome, with the help of Spt4.   References Santos-Rosa, H., Schneider, R., Bannister, A. J., Sherriff, J., Bernstein, B. E., Emre, N. C. T., Schreiber, S. L., Mellor, J., & Kouzarides, T. (2002). Active genes are tri-methylated at K4 of histone H3. Nature, 419(6905), 407–411. https://doi.org/10.1038/nature01080 Morillon, A., O'Sullivan, J., Azad, A., Proudfoot, N., & Mellor, J. (2003). Regulation of Elongating RNA Polymerase II by Forkhead Transcription Factors in Yeast. Science, 300(5618), 492–495. https://doi.org/10.1126/science.1081379 Morillon, A., Karabetsou, N., O'Sullivan, J., Kent, N., Proudfoot, N., & Mellor, J. (2003). Isw1 Chromatin Remodeling ATPase Coordinates Transcription Elongation and Termination by RNA Polymerase II. Cell, 115(4), 425–435. https://doi.org/10.1016/S0092-8674(03)00880-8 Uzun, Ü., Brown, T., Fischl, H., Angel, A., & Mellor, J. (2021). Spt4 facilitates the movement of RNA polymerase II through the +2 nucleosomal barrier. Cell Reports, 36(13), 109755. https://doi.org/10.1016/j.celrep.2021.109755   Related Episodes Effects of Non-Enzymatic Covalent Histone Modifications on Chromatin (Yael David) Nutriepigenetics: The Effects of Diet on Behavior (Monica Dus) Epigenetic Origins Of Heterogeneity And Disease (Andrew Pospisilik)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

PaperPlayer biorxiv neuroscience
Single-cell profiling of histone modifications in the mouse brain

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Sep 3, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.02.279703v1?rss=1 Authors: Bartosovic, M., Kabbe, M., Castelo-Branco, G. Abstract: The development of the mouse central nervous system (CNS) involves coordinated execution of transcriptional and epigenetic programs. These programs have been extensively studied through single-cell technologies in a pursuit to characterize the underlying cell heterogeneity. However, histone modifications pose additional layers of both positive and negative regulation that defines cellular identity. Here we show that the Cut&Tag technology can be coupled with a droplet-based single cell library preparation platform to produce high quality chromatin modifications data at a single cell resolution in tens of thousands of cells. We apply single-cell Cut&Tag (scC&T) to probe histone modifications characteristic of active promoters (H3K4me3), active promoters and enhancers (H3K27ac), active gene bodies (H3K36me3) and inactive regions (H3K27me3) and generate scC&T profiles for almost 50,000 cells. scC&T profiles of each of these histone modifications were sufficient to determine cell identity and deconvolute at single cell level regulatory principles such as promoter bivalency, spreading of H3K4me3 and promoter-enhancer connectivity. Moreover, we used scC&T to investigate the single-cell chromatin occupancy of transcription factor Olig2 and the cohesin complex component Rad21. Our results indicate that analysis of histone modifications and transcription factor occupancy at a single cell resolution can provide unique insights of epigenomic landscapes in the CNS. We also provide an online resource that can be used to interactively explore the data at https://castelobranco.shinyapps.io/BrainCutAndTag2020/. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv neuroscience
SETD1B controls cognitive function via cell type specific regulation of neuronal identity genes

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Aug 7, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.07.240853v1?rss=1 Authors: Fischer, A., Michurina, A., Sakib, M. S., Kerimoglu, C., Krüger, D. M., Kaurani, L., Islam, R., Centeno, T. P., Cha, J., Xu, X., Zeisberg, E., Kranz, A., Stewart, F. Abstract: Histone-3-lysine-4-methylation (H3K4me) is mediated by six different lysine methyltransferases (KMTs). Amongst these enzymes SET domain containing 1b (SETD1B) has been linked to intellectual disability but its role in the adult brain has not been studied yet. Here we show that mice lacking Setd1b from excitatory neurons of the adult forebrain exhibit severe memory impairment. By combining neuron-specific ChIP-seq, RNA-seq and single cell RNA-seq approaches we show that Setd1b controls the expression of neuronal-identity genes with a broad H3K4me3 peak linked to learning and memory processes. Our data furthermore suggest that basal neuronal gene-expression is ensured by other H3K4 KMTs such as Kmt2a and Kmt2b while the additional presence of Setd1b at the single cell level provides transcriptional consistency to the expression of genes important for learning & memory. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv neuroscience
Studies on C. elegans probed with estradiol, elucidate the critical role of Na+H+ exchanger, nhx-2 in reproductive senescence and neuronal health

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Jul 30, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.30.229344v1?rss=1 Authors: Shukla, S., Kumar, L., Sarkar, A., Srividya, K., Nazir, A. Abstract: Setting in of reproductive senescence (RS) gives rise to several changes, making aged individuals susceptible to neurodegenerative diseases, cardiovascular, and bone disorders amongst others. The present study deciphers the association of reproductive senescence, presence/absence of the sex hormone estradiol with age-associated neurodegenerative diseases. We employed RNAi induced silencing of a subset of 22 genes that are known to delay RS, followed by studies on alpha-Synuclein aggregation and associated effects in the transgenic C. elegans. These studies led us to functional characterisation of the Na+/H+ exchanger, expressed exclusively in the gut. We found that RNAi of nhx-2 ameliorates the effects associated with alpha-Synuclein aggregation via mimicking dietary restriction as it alters food absorption from the gut. Our studies further elucidated that such effects are Sir-2.1 driven as nhx-2 RNAi did not delay reproductive senescence when sir-2.1 was silenced concurrently. As estradiol plays a central role in both reproductive health as well as neuronal health, we performed structural binding analysis that demonstrated the binding potential of the estradiol receptor NHR-14 with nhx-2 gene. Hence, we treated the worms with estradiol and observed that the transcription levels of nhx-2 were elevated above the endogenous level. To unravel the underlying molecular mechanism of induction we performed ChIP analysis and it revealed that estradiol treatment gives rise to enhanced NHX-2 levels through inducing the promoter-specific histone H3 acetylation (H3K9) and lysine methylation (H3K4me3). Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv bioinformatics
DeepG4 : A deep learning approach to predict active G-quadruplexes

PaperPlayer biorxiv bioinformatics

Play Episode Listen Later Jul 23, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.22.215699v1?rss=1 Authors: Rocher, V., Genais, M., Nassereddine, E., Mourad, R. Abstract: DNA is a complex molecule carrying the instructions an organism needs to develop, live and reproduce. In 1953, Watson and Crick discovered that DNA is composed of two chains forming a double-helix. Later on, other structures of DNA were discovered and shown to play important roles in the cell, in particular G-quadruplex (G4). Following genome sequencing, several bioinformatic algorithms were developed to map G4s in vitro based on a canonical sequence motif, G-richness and G-skewness or alternatively sequence features including k-mers, and more recently deep learning. Here, we propose a novel convolutional neural network (DeepG4) to map active G4s (forming both in vitro and in vivo). DeepG4 is very accurate to predict active G4s, while most state-of-the-art algorithms fail. Moreover, DeepG4 identifies key DNA motifs that are predictive of G4 activity. We found that active G4 motifs do not follow a very flexible sequence pattern as current algorithms seek for. Instead, active G4s are determined by numerous specific motifs. Moreover, among those motifs, we identified known transcription factors which could play important roles in G4 activity by contributing either directly to G4 structures themselves or indirectly by participating in G4 formation in the vicinity. Lastly, variant analysis suggests that SNPs altering predicted G4 activity could affect transcription and chromatin, e.g. gene expression, H3K4me3 mark and DNA methylation. Thus, DeepG4 paves the way for future studies assessing the impact of known disease-associated variants on DNA secondary structure by providing a mechanistic interpretation of SNP impact on transcription and chromatin. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv neuroscience
A primed immune transcriptional program is activated in oligodendroglia in multiple sclerosis

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Jul 22, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.21.213876v1?rss=1 Authors: Meijer, M., Agirre, E., Kabbe, M., van Tuijn, C., Heskol, A., Falcao, A. M., Corces, M. R., Montine, T., Chen, X., Chang, H. Y., Castelo-Branco, G. Abstract: Multiple sclerosis (MS) is a disease characterized by a targeted immune attack on myelin in the central nervous system (CNS). We have previously shown that oligodendrocytes (OLs), myelin producing cells in the CNS, and their precursors (OPCs), acquire disease-specific transcriptional states in MS1,2. To understand how these alternative transcriptional programs are activated in disease, we performed single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) on the OL lineage in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We identified regulatory regions with increased accessibility in oligodendroglia (OLG) in EAE, some of which in the proximity of immune genes. A similar remodeling of chromatin accessibility was observed upon treatment of postnatal OPCs with interferon-gamma (IFN-gamma), but not with dexamethasone. These changes in accessibility were not exclusive to distal enhancers, but also occurred at promoter regions, suggesting a role for promoters in mediating cell-state transitions. Notably, we found that a subset of immune genes already exhibited chromatin accessibility in OPCs ex vivo and in vivo, suggesting a primed chromatin state in OLG compatible with rapid transitions to an immune-competent state. Several primed genes presented bivalency of H3K4me3 and H3K27me3 at promoters in OPCs, with loss of H3K27me3 upon IFN-gamma treatment. Inhibition of JMJD3/Kdm6b, mediating removal of H3K27me3, led to the inability to activate these genes upon IFN-gamma treatment. Importantly, OLGs from the adult human brain showed chromatin accessibility at immune gene loci, particularly at MHC-I pathway genes. A subset of single-nucleotide polymorphisms (SNPs) associated with MS susceptibility overlapped with these primed regulatory regions in OLG from both mouse and human CNS. Our data suggest that susceptibility for MS may involve activation of immune gene programs in OLG. These programs are under tight control at the chromatin level in OLG and may therefore constitute novel targets for immunological-based therapies for MS. Copy rights belong to original authors. Visit the link for more info

PaperPlayer biorxiv neuroscience
Altered Huntingtin-Chromatin Interactions Predict Transcriptional and Epigenetic Changes in Huntingtons Disease

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Jun 5, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.04.132571v1?rss=1 Authors: Pearl, J. R., Shetty, A. C., Cantle, J. P., Bergey, D. E., Bragg, R. M., Coffey, S. R., Kordasiewicz, H. B., Hood, L. E., Price, N. D., Ament, S. A., Carroll, J. B. Abstract: Progressive striatal gene expression changes and epigenetic alterations are a prominent feature of Huntingtons disease (HD), but direct relationships between the huntingtin (HTT) protein and chromatin remain poorly described. Here, using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that HTT reproducibly occupies specific locations in the mouse genome, including thousands of genomic loci that are differentially occupied in striatal tissue from a knock-in mouse model of HD (B6.HttQ111/+) versus wildtype controls. ChIP-seq of histone modifications, generated in parallel, revealed genotype-specific colocalization of HTT with trimethylation of histone 3 lysine 27 (H3K27me3), a repressive chromatin mark. Close to genes that are differentially regulated in HD, greater HTT occupancy in HttQ111/+ vs. wildtype mice predicted increased H3K27me3, reduced histone 3 lysine 4 (H3K4me3, a marker of poised and active promoters), and down-regulated gene expression. Altered huntingtin-chromatin interactions may therefore play a direct role in driving transcriptional dysregulation in HD. Copy rights belong to original authors. Visit the link for more info

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06
Stage-dependent changes of the nuclear architecture, envelope and lamina during mammalian early embryonic development studied with a novel 3D structured illumination microscopy protocol

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06

Play Episode Listen Later Jan 15, 2016


Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) features an 8-fold volumetric resolution improvement over conventional microscopy and is well established on flat, adherent cells. However, blastomeres in mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to non-adherent embryos moving during scanning and a large distance to the cover glass. The biggest challenge and achievement of this doctorate thesis was the development of a novel method to perform 3D-SIM on mammalian embryos (“3D structured illumination microscopy of mammalian embryos and spermatozoa” published in BMC Developmental Biology). The development and fine-tuning of this method took over two years due to the time-intense generation of embryos and the subsequent two day long embryo staining, embedding and scanning with steps that required novel techniques such as micromanipulation which was not associated with sample preparation prior to this protocol. Problem identification was time-intensive since each of the numerous steps necessary could negatively affect the image quality. This method was fine-tuned during three studies. The first study “Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos” (published in Nucleus) investigates the profound changes of nuclear architecture during cattle preimplantation development of embryos generated by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Fibroblast nuclei in embryos generated by SCNT go through similar changes in nuclear architecture as embryos generated by IVF. In both embryo types the occurrence of a large, chromatin-free lacuna in the center of nuclei around major embryonic genome activation (EGA) was noted. Similarly, the chromosome territory-interchromatin compartment (CT-IC) model applied to both types of embryos, featuring a lacuna or not, with an enrichment of RNA polymerase II and H3K4me3, a histone modification for transcriptionally competent chromatin, in less concentrated chromatin and an enrichment of H3K9me3, a transcriptionally restrictive histone modification, in more concentrated chromatin. However, large, highly concentrated H3K4me3 and H3K9me3 clusters were noted in both embryo types at chromatin concentrations that did not fit to the model. The chromatin-free lacunas were highly enriched in newly synthesized mRNA. The second study “Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications” (published in PLOS ONE) presents the changes of the nuclear envelope and lamina during bovine preimplantation development. Before major EGA, chromatin-free areas of the nuclear periphery were also free of nuclear pore complexes (NPCs), whereas after major EGA, the entire nuclear periphery was equipped with at least a fine layer of chromatin and associated NPCs. Three types of nuclear invaginations were predominant at different stages. The most common invagination was lamin B and NUP153 positive and was most prominent between the 2-cell and 8-cell stages until the onset of major EGA. Lamin B positive, but NUP153 negative invaginations were most prominent during stages with large nuclear volume and surface reductions. The least common invagination was lamin B negative but NUP153 positive and occurred almost exclusively at the morula stage. RNA-Seq and 3D-SIM data showed large deposits of spliced NUP153 mRNA and cytoplasmic NUP153 protein clusters until shortly after major EGA. NUP153 association with chromatin was initiated at metaphase. The third study “Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development” (published in the Journal of Reproduction and Development) demonstrated that rabbit embryonic nuclei feature a nuclear invagination type containing a large volume of cytoplasm that provides cytoplasmic proximity to nucleoli in addition to the small volume invaginations that were previously observed in bovine nuclei. The underlying mechanism for these two invaginations must differ from each other since small volume invaginations were frequently emanating from large volume invaginations emanating from the nuclear border but large volume invaginations were never emanating from small volume invaginations emanating from the nuclear border. Abundance of import/export competent invaginations featuring NPCs peaked at the 4-cell stage, which is the last stage before a drastic nuclear volume decline and also the last stage before major EGA is initiated at the 8- to 16-cell stage. Import/export incompetent invaginations positive for lamin B but not NUP153 peaked at the 2-cell stage. This was the stage with the largest variability in nuclear volumes. This may hint at an interphase nuclear surface reduction mechanism. Additionally, previously generated but unpublished 3D-FISH data about the localization changes of a stably inserted reporter gene upon activation in cloned bovine embryos was analyzed and documented in the study “Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos” (published in Nucleus). This study showed that the stably inserted OCT-4 reporter gene “GOF” in bovine fetal fibroblasts was initially moved towards the nuclear interior in day 2 bovine embryos generated by SCNT of bovine fetal fibroblasts. However, in day 4 SCNT embryos the localization of GOF had moved towards the periphery while it was still activated. Its carrier chromosome territory did not significantly move differently compared with the non-carrier homolog. Constant proximity of GOF to its carrier chromosome territory ruled out a movement by giant loops. In cooperation with the Department of Histology and Embryology of the Ege University (Izmir, Turkey) the destructive effects of cryopreservation on blastomere integrity were analyzed in the study “Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification” (published in Anatomia, Histologia, Embryologia). The cryopreservation method slow freezing caused more damage to blastomeres and to the zona pellucida than its fast freezing alternative vitrification. This was most likely caused by ice crystal formation and the longer exposure to the toxic side effects of cryoprotectants before freezing was complete.

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06
Prevention and prediction of production instability of CHO-K1 cell lines by the examination of epigenetic mechanisms

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06

Play Episode Listen Later Oct 27, 2015


The CHO-K1 cell line is the most common expression system for therapeutic proteins in the pharmaceutical industry. Due to the nature of economics, the cell lines and the vector design are subject to constant change to increase product quality and quantity. During the cultivation, the production cell lines are susceptible to decreasing productivity over time. Often the loss of production can be associated with a reduction of copy number and the silencing of transgenes. During cell line development, the most promising cell lines are cultivated in large batch culture. Consequently, the loss of a stable production cell line can be very cost-intensive. For this reason I developed different strategies to avoid a reduced productivity. Instability of production cell lines can be predicted by the degree of CpG methylation of the driving promoter. Considering that the DNA methylation is at the end of an epigenetic cascade and associated with the maintenance of the repressive state, I investigated the upstream signals of histone modifications with the assumption to obtain a higher predictive power of production instability. For this reason I performed a chromatin immunoprecipitation of the histone modifications H3K9me3 and H3K27me3 as repressive signals and H3ac as well as H3K4me3 as active marks. The accumulations of those signals were measured close to the hCMV-MIE at the beginning of the cultivation and were then compared with the loss of productivity over two month. I found that the degree of the H3 acetylation (H3ac) correlated best with the production stability. Furthermore I was able to identify an H3ac threshold to exclude most of the unstable producers. In the second project I aimed to improve the vector design by considering epigenetic mechanisms. To this end I designed on the one hand a target-oriented histone acetyltransferase to enforce an open and active chromatin status at the transgene. On the other hand I point-mutated methylation-susceptible CpGs within the hCMV-MIE to impede the maintenance of inactive heterochromatin formation. Remarkably, the C to G mutation located 179 bp upstream of transcription start site resulted in very stable antibody producing cell lines. In addition, the examination of cell pools expressing eGFP showed that G-179 promoter variants were less prone to a general methylation and gene amplification, which illustrates the dominating effect in epigenetic mechanisms of one single CpG. The last project was performed to localize stable integration sites within the CHO-K1 genome. In so doing I could show that the transfection leads predominantly to integration into inactive regions. Furthermore I identified promising integration sites with a high potential to induce stable expression. However, those results are preliminary and must be viewed with caution. Further examination needs to be done to confirm these results. Considering the results of all three projects, I propose that the interplay of metabolic burden and selection pressure at an early time point of cultivation plays an important role in cell line development. Small alterations of selection pressure can lead to a decisive change of cell properties. Therefore, stable cells are less susceptible than weak producers. The increase of selection pressure leads to compensatory effect by gene amplification in the instable cell lines. The resulting adjustment of productivity masks the truly stable cells, which precludes the selection of the right cell lines. For this reason the selection pressure, the copy number as well as the growth rate should be considered to minimize repressive effects.

Medizin - Open Access LMU - Teil 22/22
Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines

Medizin - Open Access LMU - Teil 22/22

Play Episode Listen Later Jan 1, 2014


Background: Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. Methods: A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. Results: The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual gamma-H2AX foci after 24 h. Conclusion: So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.

Medizin - Open Access LMU - Teil 18/22
H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation

Medizin - Open Access LMU - Teil 18/22

Play Episode Listen Later Mar 1, 2011


Methylation of histone H3 lysine 4 (H3K4me) is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac) has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs) Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC) Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP), we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3), a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS), which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1D mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me).

Medizin - Open Access LMU - Teil 15/22
Evidence for conserved DNA and histone H3 methylation reprogramming in mouse, bovine and rabbit zygotes

Medizin - Open Access LMU - Teil 15/22

Play Episode Listen Later Jan 1, 2008


Background: In mammals the parental genomes are epigenetically reprogrammed after fertilization. This reprogramming includes a rapid demethylation of the paternal (sperm-derived) chromosomes prior to DNA replication in zygotes. Such active DNA demethylation in the zygote has been documented for several mammalian species, including mouse, rat, pig, human and cow, but questioned to occur in rabbit. Results: When comparing immunohistochemical patterns of antibodies against 5-methyl-cytosine, H3K4me3 and H3K9me2 modifications we observe similar pronuclear distribution and dynamics in mouse, bovine and rabbit zygotes. In rabbit DNA demethylation of the paternal chromosomes occurs at slightly advanced pronuclear stages. We also show that the rabbit oocyte rapidly demethylates DNA of donor fibroblast after nuclear transfer. Conclusion: Our data reveal that major events of epigenetic reprogramming during pronuclear maturation, including mechanisms of active DNA demethylation, are apparently conserved among mammalian species.