scholarly journals Spreading and epigenetic inheritance of heterochromatin require a critical density of histone H3 lysine 9 tri-methylation

2021 ◽  
Vol 118 (22) ◽  
pp. e2100699118
Author(s):  
Amber R. Cutter DiPiazza ◽  
Nitika Taneja ◽  
Jothy Dhakshnamoorthy ◽  
David Wheeler ◽  
Sahana Holla ◽  
...  
Keyword(s):  
Histone H3 ◽  
Critical Density ◽  
Epigenetic Inheritance ◽  
Dominant Negative ◽  
Yeast Genome ◽  
Stable Gene ◽  
Dependent Manner ◽  
Histone Methyltransferases ◽  
Dominant Negative Mutation ◽  
Heterochromatin Assembly

Heterochromatin assembly requires methylation of histone H3 lysine 9 (H3K9me) and serves as a paradigm for understanding the importance of histone modifications in epigenetic genome control. Heterochromatin is nucleated at specific genomic sites and spreads across extended chromosomal domains to promote gene silencing. Moreover, heterochromatic structures can be epigenetically inherited in a self-templating manner, which is critical for stable gene repression. The spreading and inheritance of heterochromatin are believed to be dependent on preexisting H3K9 tri-methylation (H3K9me3), which is recognized by the histone methyltransferase Clr4/Suv39h via its chromodomain, to promote further deposition of H3K9me. However, the process involving the coupling of the “read” and “write” capabilities of histone methyltransferases is poorly understood. From an unbiased genetic screen, we characterize a dominant-negative mutation in histone H3 (H3G13D) that impairs the propagation of endogenous and ectopic heterochromatin domains in the fission yeast genome. H3G13D blocks methylation of H3K9 by the Clr4/Suv39h methyltransferase and acts in a dosage-dependent manner to interfere with the spreading and maintenance of heterochromatin. Our analyses show that the incorporation of unmethylatable histone H3G13D into chromatin decreases H3K9me3 density and thereby compromises the read-write capability of Clr4/Suv39h. Consistently, enhancing the affinity of Clr4/Suv39h for methylated H3K9 is sufficient to overcome the defects in heterochromatin assembly caused by H3G13D. Our work directly implicates methylated histones in the transmission of epigenetic memory and shows that a critical density threshold of H3K9me3 is required to promote epigenetic inheritance of heterochromatin through the read-write mechanism.

scholarly journals COP9 signalosome is an essential and druggable parasite target that regulates protein degradation

2020 ◽  
Author(s):  
Swagata Ghosh ◽  
Laura Farr ◽  
Aditya Singh ◽  
Laura-Ann Leaton ◽  
Jay Padalia ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Therapeutic Potential ◽  
Degradation Pathway ◽  
Dominant Negative ◽  
Cop9 Signalosome ◽  
Dependent Manner ◽  
Parasite Protein ◽  
Upstream Regulator ◽  
Dominant Negative Mutation

ABSTRACTUnderstanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved alcohol-abuse drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition.Summary sentenceParasite-encoded COP9 signalosome is an essential upstream regulator of ubiquitin-proteasome mediated protein degradation, and shows significant potential as a therapeutic target.

scholarly journals An RNA Degradation Complex Required for Spreading and Epigenetic Inheritance of Heterochromatin

2019 ◽  
Author(s):  
Gergana Shipkovenska ◽  
Alexander Durango ◽  
Marian Kalocsay ◽  
Steven P. Gygi ◽  
Danesh Moazed
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Rna ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Rna Degradation ◽  
Rrna Processing ◽  
Nucleation Sites ◽  
Polynucleotide Kinase ◽  
Heterochromatin Assembly

AbstractHeterochromatin assembly requires the methylation of histone H3 lysine 9 by the Clr4(Suv39h) methyltransferase and both the spreading and epigenetic inheritance of heterochromatin involve the recognition of H3K9me-containing nucleosomes by Clr4 and catalysis of H3K9me on adjacent nucleosomes. How this read-write mechanism overcomes obstacles posed by RNA polymerase II and nascent RNA in its path is not fully understood. Here we identify a role for the highly conserved and essential Rix1-containing complex (here referred to as the rixosome), with known RNA endonuclease and polynucleotide kinase activities required for ribosomal RNA (rRNA) processing, in spreading and epigenetic inheritance of heterochromatin. Viable mutations in rixosome subunits that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing away from nucleation sites into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.

How do histone modifications contribute to transgenerational epigenetic inheritance in C. elegans?

2020 ◽  
Vol 48 (3) ◽  
pp. 1019-1034 ◽  
Author(s):  
Rachel M. Woodhouse ◽  
Alyson Ashe
Keyword(s):  
Histone Modifications ◽  
Molecular Mechanisms ◽  
Epigenetic Inheritance ◽  
Nematode Caenorhabditis Elegans ◽  
Histone Methyltransferases ◽  
Transgenerational Epigenetic Inheritance ◽  
C Elegans ◽  
Recent Developments ◽  
Gene Regulatory

Gene regulatory information can be inherited between generations in a phenomenon termed transgenerational epigenetic inheritance (TEI). While examples of TEI in many animals accumulate, the nematode Caenorhabditis elegans has proven particularly useful in investigating the underlying molecular mechanisms of this phenomenon. In C. elegans and other animals, the modification of histone proteins has emerged as a potential carrier and effector of transgenerational epigenetic information. In this review, we explore the contribution of histone modifications to TEI in C. elegans. We describe the role of repressive histone marks, histone methyltransferases, and associated chromatin factors in heritable gene silencing, and discuss recent developments and unanswered questions in how these factors integrate with other known TEI mechanisms. We also review the transgenerational effects of the manipulation of histone modifications on germline health and longevity.

scholarly journals The Role of Alcohol, LPS Toxicity, and ALDH2 in Dental Bony Defects

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 651
Author(s):  
Hsiao-Cheng Tsai ◽  
Che-Hong Chen ◽  
Daria Mochly-Rosen ◽  
Yi-Chen Ethan Li ◽  
Min-Huey Chen
Keyword(s):  
Bone Loss ◽  
Bacterial Infection ◽  
Bone Regeneration ◽  
Alcohol Drinking ◽  
Bacterial Species ◽  
Dominant Negative ◽  
Enzyme Inactivation ◽  
Wild Type ◽  
Micro Computed Tomography ◽  
Dominant Negative Mutation

It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.

scholarly journals Cellular Werner Phenotypes in Mice Expressing a Putative Dominant-Negative Human WRN Gene

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 357-362
Author(s):  
Lan Wang ◽  
Charles E Ogburn ◽  
Carol B Ware ◽  
Warren C Ladiges ◽  
Hagop Youssoufian ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mice ◽  
Werner Syndrome ◽  
Dominant Negative ◽  
Dominant Negative Mutation ◽  
Fibroblast Cultures ◽  
Age Related

Abstract Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS). WS patients prematurely develop an aged appearance and various age-related disorders. We have generated transgenic mice expressing human WRN with a putative dominant-negative mutation (K577M-WRN). Primary tail fibroblast cultures from K577M-WRN mice showed three characteristics of WS cells: hypersensitivity to 4-nitroquinoline-1-oxide (4NQO), reduced replicative potential, and reduced expression of the endogenous WRN protein. These data suggest that K577M-WRN mice may provide a novel mouse model for the WS.

scholarly journals The new kid on the block: A dominant‐negative mutation of phototropin1 enhances carotenoid content in tomato fruits

2021 ◽  
Author(s):  
Himabindu Vasuki Kilambi ◽  
Alekhya Dindu ◽  
Kapil Sharma ◽  
Narasimha Rao Nizampatnam ◽  
Neha Gupta ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Carotenoid Content ◽  
Tomato Fruits ◽  
Dominant Negative Mutation

scholarly journals With-No-Lysine Kinase 1 (WNK1) Augments TRPV4 Function in the Aldosterone-Sensitive Distal Nephron

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1482
Author(s):  
Viktor N. Tomilin ◽  
Kyrylo Pyrshev ◽  
Naghmeh Hassanzadeh Khayyat ◽  
Oleg Zaika ◽  
Oleh Pochynyuk
Keyword(s):  
Collecting Duct ◽  
Chinese Hamster ◽  
Dominant Negative ◽  
K Channel ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Distal Nephron ◽  
Potassium Homeostasis ◽  
Wnk Kinases ◽  
K Secretion

Kidneys play a central role in regulation of potassium homeostasis and maintenance of plasma K+ levels within a narrow physiological range. With-no-lysine (WNK) kinases, specifically WNK1 and WNK4, have been recognized to regulate K+ balance, in part, by orchestrating maxi K+ channel (BK)-dependent K+ secretion in the aldosterone-sensitive distal nephron (ASDN), which includes the connecting tubule and collecting duct. We recently demonstrated that the Ca2+-permeable TRPV4 channel is essential for BK activation in the ASDN. Furthermore, high K+ diet increases TRPV4 activity and expression largely in an aldosterone-dependent manner. In the current study, we aimed to test whether WNK kinases contribute to regulation of TRPV4 activity and its stimulation by aldosterone. Systemic inhibition of WNK with WNK463 (1 mg/kgBW for 3 days) markedly decreased TRPV4-dependent Ca2+ influx in freshly isolated split-opened collecting ducts. Aldosterone greatly increased TRPV4 activity and expression in cultured mpkCCDc14 cells and this effect was abolished in the presence of WNK463. Selective inhibition of WNK1 with WNK-in-11 (400 nM, 24 h) recapitulated the effects of WNK463 on TRPV4-dependent Ca2+ influx. Interestingly, WNK-in-11 did not interfere with up-regulation of TRPV4 expression by aldosterone, but prevented translocation of the channel to the apical plasma membrane. Furthermore, co-expression of TRPV4 and WNK1 into Chinese hamster ovary (CHO) cells increased the macroscopic TRPV4-dependent cation currents. In contrast, over-expression of TRPV4 with a dominant negative WNK1 variant (K233M) decreased the whole-cell currents, suggesting both stimulatory and permissive roles of WNK1 in regulation of TRPV4 activity. Overall, we show that WNK1 is essential for setting functional TRPV4 expression in the ASDN at the baseline and in response to aldosterone. We propose that this new mechanism contributes to regulation of K+ secretion and, by extension, urinary K+ levels to maintain systemic potassium homeostasis.

scholarly journals Fibroblast Growth Factors Regulate Prolactin Transcription via an Atypical Rac-Dependent Signaling Pathway

2003 ◽  
Vol 17 (10) ◽  
pp. 1921-1930 ◽  
Author(s):  
Twila A. Jackson ◽  
David M. Koterwas ◽  
Melissa A. Morgan ◽  
Andrew P. Bradford
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Signaling Pathway ◽  
Promoter Activity ◽  
Fibroblast Growth Factors ◽  
Dominant Negative ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
Prl Gene

Abstract Fibroblast growth factors (FGFs) play a critical role in pituitary development and in pituitary tumor formation and progression. We have previously characterized FGF signal transduction and regulation of the tissue-specific rat prolactin (rPRL) promoter in GH4 pituitary cells. FGF induction of rPRL transcription is independent of Ras, but mediated by a protein kinase C-δ (PKCδ)-dependent activation of MAPK (ERK). Here we demonstrate a functional role for the Rho family monomeric G protein, Rac1, in FGF regulation of PRL gene expression via an atypical signaling pathway. Expression of dominant negative Rac, but not RhoA or Cdc42, selectively inhibited FGF-induced rPRL promoter activity. Moreover, expression of dominant negative Rac also attenuated FGF-2 and FGF-4 stimulation of MAPK (ERK). However, in contrast to other Rac-dependent signaling pathways, FGF activation of rPRL promoter activity was independent of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase/Akt cascades. FGFs failed to activate JNK1 or JNK2, and expression of dominant negative JNK or Akt constructs did not block FGF-induced PRL transcription. Consistent with the role of PKCδ in FGF regulation of PRL gene expression, activation of the rPRL promoter was blocked by an inhibitor of phospholipase Cγ (PLCγ) activity. FGF treatment also induced rapid tyrosine phosphorylation of PLCγ in a Rac-dependent manner. These results suggest that FGF-2 and FGF-4 activate PRL gene expression via a novel Rac1, PLCγ, PKCδ, and ERK cascade, independent of phosphoinositol-3-kinase and JNK.

scholarly journals Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fosPromoter

2000 ◽  
Vol 20 (4) ◽  
pp. 1140-1148 ◽  
Author(s):  
Dae-Won Kim ◽  
Brent H. Cochran
Keyword(s):  
Map Kinase ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Binding Motif ◽  
Dependent Manner ◽  
Consensus Map ◽  
Interaction Domain ◽  
Extracellular Signal

ABSTRACT We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fospromoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

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