scholarly journals MBD5 and MBD6 couple DNA methylation to gene silencing through the J-domain protein SILENZIO

Science ◽  
2021 ◽  
pp. eabg6130
Author(s):  
Lucia Ichino ◽  
Brandon A. Boone ◽  
Luke Strauskulage ◽  
C. Jake Harris ◽  
Gundeep Kaur ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Gene Silencing ◽  
Molecular Chaperone ◽  
Transcriptional Repression ◽  
Transcriptional Repressor ◽  
Loss Of Function ◽  
Eukaryotic Genes ◽  
J Domain ◽  
Domain Protein

DNA methylation is associated with transcriptional repression of eukaryotic genes and transposons, but the downstream mechanism of gene silencing is largely unknown. Here we describe two Arabidopsis methyl-CpG binding domain proteins, MBD5 and MBD6, that are recruited to chromatin by recognition of CG methylation, and redundantly repress a subset of genes and transposons without affecting DNA methylation levels. These methyl-readers recruit a J-domain protein, SILENZIO, that acts as a transcriptional repressor in loss-of-function and gain-of-function experiments. J-domain proteins often serve as co-chaperones with HSP70s. Indeed, we found that SILENZIO’s conserved J-domain motif was required for its interaction with HSP70s and for its silencing function. These results uncover an unprecedented role of a molecular chaperone J-domain protein in gene silencing downstream of DNA methylation.

scholarly journals Limited consequences for loss of RNA-directed DNA methylation in Setaria viridis domains rearranged methyltransferase (DRM) mutants

2022 ◽  
Author(s):  
Andrew C. Read ◽  
Trevor Weiss ◽  
Peter A. Crisp ◽  
Zhikai Liang ◽  
Jaclyn Noshay ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcriptional Repression ◽  
Regulation Of Gene Expression ◽  
Transcriptome Profiling ◽  
Setaria Viridis ◽  
Loss Of Function ◽  
Promoter Regions ◽  
Altered Expression ◽  
Genome Methylation

The Domains Rearranged Methyltransferases (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. The analysis of drm1ab double mutant plants revealed limited morphological consequences for the loss of RdDM. Whole-genome methylation profiling provided evidence for wide-spread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. There is also evidence for locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified a limited number of genes with altered expression in the drm1ab mutants. The majority of genes with elevated CHH methylation directly surrounding the transcription start site or in nearby promoter regions do not have altered expression in the drm1ab mutant even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of transposable elements identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons likely require active RdDM for maintenance of transcriptional repression.

scholarly journals Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1187
Author(s):  
Michael Wassenegger ◽  
Athanasios Dalakouras
Keyword(s):  
Dna Methylation ◽  
Rna Interference ◽  
Gene Silencing ◽  
Plant Cells ◽  
Transcriptional Gene Silencing ◽  
Rnai Machinery ◽  
Double Stranded Rna ◽  
Post Transcriptional Gene Silencing ◽  
Cumulative Amount

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

scholarly journals Functional Domains of c-myc Promoter Binding Protein 1 Involved in Transcriptional Repression and Cell Growth Regulation

1999 ◽  
Vol 19 (4) ◽  
pp. 2880-2886 ◽  
Author(s):  
Asish K. Ghosh ◽  
Robert Steele ◽  
Ratna B. Ray
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Dna Binding ◽  
Transcriptional Repression ◽  
Growth Regulation ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Repressor Activity

ABSTRACT We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library. Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells. To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed. This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites. Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244). The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region. When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited. Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common. Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression. Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-mycpromoter. In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1. Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation.

scholarly journals Role of the Arabidopsis DRM Methyltransferases in De Novo DNA Methylation and Gene Silencing

2002 ◽  
Vol 12 (13) ◽  
pp. 1138-1144 ◽  
Author(s):  
Xiaofeng Cao ◽  
Steven E. Jacobsen
Keyword(s):  
Dna Methylation ◽  
Gene Silencing ◽  
De Novo

scholarly journals Inhibition of a transcriptional repressor rescues hearing in a splicing factor–deficient mouse

2020 ◽  
Vol 3 (12) ◽  
pp. e202000841
Author(s):  
Yoko Nakano ◽  
Susan Wiechert ◽  
Bernd Fritzsch ◽  
Botond Bánfi
Keyword(s):  
Alternative Splicing ◽  
Target Genes ◽  
Transcriptional Repressor ◽  
Dominant Negative ◽  
Splicing Factor ◽  
Loss Of Function ◽  
Transgenic Expression ◽  
Mechanosensory Hair Cells

In mechanosensory hair cells (HCs) of the ear, the transcriptional repressor REST is continuously inactivated by alternative splicing of its pre-mRNA. This mechanism of REST inactivation is crucial for hearing in humans and mice. Rest is one of many pre-mRNAs whose alternative splicing is regulated by the splicing factor SRRM4; Srrm4 loss-of-function mutation in mice (Srrm4bv/bv) causes deafness, balance defects, and degeneration of all HC types other than the outer HCs (OHCs). The specific splicing alterations that drive HC degeneration in Srrm4bv/bv mice are unknown, and the mechanism underlying SRRM4-independent survival of OHCs is undefined. Here, we show that transgenic expression of a dominant-negative REST fragment in Srrm4bv/bv mice is sufficient for long-term rescue of hearing, balancing, HCs, alternative splicing of Rest, and expression of REST target genes including the Srrm4 paralog Srrm3. We also show that in HCs, SRRM3 regulates many of the same exons as SRRM4; OHCs are unique among HCs in that they transiently down-regulate Rest transcription as they mature to express Srrm3 independently of SRRM4; and simultaneous SRRM4–SRRM3 deficiency causes complete HC loss by preventing inactivation of REST in all HCs. Thus, our data reveal that REST inactivation is the primary and essential role of SRRM4 in the ear, and that OHCs differ from other HCs in the SRRM4-independent expression of the functionally SRRM4-like splicing factor SRRM3.

scholarly journals Domain-Specific Response of Imprinted Genes to Reduced DNMT1

2010 ◽  
Vol 30 (16) ◽  
pp. 3916-3928 ◽  
Author(s):  
Jamie R. Weaver ◽  
Garnik Sarkisian ◽  
Christopher Krapp ◽  
Jesse Mager ◽  
Mellissa R. W. Mann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Critical Role ◽  
Imprinted Genes ◽  
Specific Response ◽  
Loss Of Function ◽  
Partial Loss ◽  
Domain Specific ◽  
Parent Of Origin

ABSTRACT Imprinted genes are expressed in a monoallelic, parent-of-origin-specific manner. Clusters of imprinted genes are regulated by imprinting control regions (ICRs) characterized by DNA methylation of one allele. This methylation is critical for imprinting; a reduction in the DNA methyltransferase DNMT1 causes a widespread loss of imprinting. To better understand the role of DNA methylation in the regulation of imprinting, we characterized the effects of Dnmt1 mutations on the expression of a panel of imprinted genes in the embryo and placenta. We found striking differences among imprinted domains. The Igf2 and Peg3 domains showed imprinting perturbations with both null and partial loss-of-function mutations, and both domains had pairs of coordinately regulated genes with opposite responses to loss of DNMT1 function, suggesting these domains employ similar regulatory mechanisms. Genes in the Kcnq1 domain were less sensitive to the absence of DNMT1. Cdkn1c exhibited imprinting perturbations only in null mutants, while Kcnq1 and Ascl2 were largely unaffected by a loss of DNMT1 function. These results emphasize the critical role for DNA methylation in imprinting and reveal the different ways it controls gene expression.

scholarly journals The J domain of sacsin disrupts intermediate filament assembly

2021 ◽  
Author(s):  
Afrooz Dabbaghizadeh ◽  
Alexandre Pare ◽  
Zacharie Cheng-Boivin ◽  
Robin Dagher ◽  
Sandra Minotti ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Loss Of Function ◽  
Cultured Fibroblasts ◽  
Filament Assembly ◽  
A Cell ◽  
J Domain ◽  
Initial Assembly ◽  
In Cells

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS), is caused by loss of function mutations in the SACS gene, which encodes sacsin, a giant protein of 520 kDa. A key feature of the absence of sacsin in cells is the formation of abnormal bundles of intermediate filaments (IF) including neurofilaments (NF) in neurons and vimentin IF in fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs-/- neurons, however, its mechanism is still unclear. Here, we focused on the role of SacsJ in NF assembly. We report that the SacsJ domain directly interacts with NF proteins in vitro to disassemble NFL filaments, and to inhibit their initial assembly, in the absence of Hsp70. We generated a cell-penetrating peptide derived from this domain, SacsJ-myc-TAT, which was efficient in disassembling both vimentin IF and NF in cultured fibroblasts and Sacs+/+ motor neurons as well as NF bundles in cultured Sacs-/- motor neurons. Whereas a normal NF network was restored in Sacs-/- neurons treated with the SacsJ peptide, there was some loss of IF networks in Sacs+/+ fibroblasts or neurons. These results suggest that SacsJ is a key regulator of NF and IF networks in cells, with implications for its therapeutic use.

scholarly journals Brassinosteroid signaling component SlBES1 promotes tomato fruit softening through transcriptional repression of PMEU1

2021 ◽  
Author(s):  
Haoran Liu ◽  
Lihong Liu ◽  
Dongyi Liang ◽  
Min Zhang ◽  
Chengguo Jia ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Tomato Fruit ◽  
Fruit Softening ◽  
Loss Of Function ◽  
Postharvest Storage ◽  
Nutrition Quality ◽  
Transcriptional Inhibition ◽  
Tomato Fruit Ripening ◽  
Brassinosteroid Signaling

ABSTRACTFirmness is one of the most important factors that affect postharvest properties of tomato fruit. However, the regulatory mechanism underlying firmness formation in tomato fruit is poorly understood. Here, we report a novel role of SlBES1, a transcriptional factor (TF) mediating brassinosteroid (BR) signaling, in tomato fruit softening. We first found that SlBES1 promotes fruit softening during tomato fruit ripening and postharvest storage. RNA-seq analysis suggested that PMEU1, which encodes a pectin de-methylesterification protein, might participate in SlBES1-mediated fruit softening. Biochemical and immunofluorescence assays in SlBES1 transgenic fruits indicated that SlBES1 inhibited PMEU1-related pectin de-methylesterification. Further molecular and genetic evidence verified that SlBES1 directly binds to the E-box in the promoter of PMEU1 to repress its expression, leading to the softening of the tomato fruits. Loss-of-function SlBES1 mutant generated by CRISPR/cas9 showed firmer fruits and longer shelf life during postharvest storage without the color, size and nutritional quality alteration. Collectively, our results indicated the potential of manipulating SlBES1 to regulate fruit firmness via transcriptional inhibition of PMEU1 without negative consequence on visual and nutrition quality.

Both the Activity and Stability of the Transcriptional Repressor PLZF Are Modified by the Class III Histone Deacetylase SIRT1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 360-360
Author(s):  
Melanie-Jane McConnell ◽  
Emma Langley ◽  
Yolanda Martinez-Martinez ◽  
Tony Kouzarides ◽  
Jonathan D. Licht
Keyword(s):  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Transcriptional Repressor ◽  
Model Organisms ◽  
Class Iii ◽  
Endogenous Proteins ◽  
Co Precipitation ◽  
Protein Deacetylase

Abstract The transcription factor PLZF is expressed in hematopoietic development and rearranged in t(11;17) acute promyelocytic leukemia (APL). PLZF expression is high in the quiescent progenitor CD34+ cell, and declines during differentiation along myeloid and erythroid lineages. PLZF encodes a BTB-Zinc finger transcriptional repressor that inhibits the cell cycle through inhibition of targets genes such as cyclin A and c-myc through the recruitment of histone deacetylase complexes. However, PLZF itself is regulated by acetylation. In a separate study we demonstrate that acetylation of PLZF by p300/CBP enhances the transcriptional repression activity of PLZF. Through a yeast two-hybrid study we found that PLZF associates with the protein deacetylase SIRT1. SIRT1 is a member of the sirtuin family of class III histone deacetylases. In model organisms such as yeast, worms and flies, sirtuins play a common role in lifespan extension. The interaction between PLZF and SIRT1 was confirmed by co-precipitation of endogenous proteins and localized to the zinc fingers of PLZF, the region targeted for acetylation by p300/CBP. Acetylation of PLZF mediated by p300/CBP was reversed by SIRT1. Furthermore while acetylation of PLZF enhances its ability to repress transcription, co-expression of SIRT1 decreased PLZF transcriptional repression activity, consistent with loss of acetylation. Conversely, inhibition of SIRT1 activity with nicotinamide enhanced both PLZF acetylation and transcriptional repression of PLZF on its endogenous target gene c-myc. Further, increasing PLZF acetylation by inhibition of SIRT1 was associated with decreased PLZF stability. Acetyl-PLZF levels were stabilized by inhibition of the proteosomal degradation machinery, together implying that PLZF acetylation and activation results in increased protein turnover. These data point to the increasing complexity of the role of acetylation in transcriptional regulation and stand in contrast with data for the related Bcl6 repressor where acetylation of the protein inhibits repression. These data also indicate a novel function for sirtuins in regulation of transcriptional repression.

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