scholarly journals Modulation of Chromatin Boundary Activities by Nucleosome-Remodeling Activities in Drosophila melanogaster

2009 ◽  
Vol 30 (4) ◽  
pp. 1067-1076 ◽  
Author(s):  
Mo Li ◽  
Vladimir E. Belozerov ◽  
Haini N. Cai
Keyword(s):  
Drosophila Melanogaster ◽  
Nucleosome Occupancy ◽  
Boundary Function ◽  
Higher Order ◽  
Chromatin Organization ◽  
Nurd Complex ◽  
Nucleosome Remodeling ◽  
Chromatin Boundary ◽  
Enhancer Blocking

ABSTRACT Chromatin boundaries facilitate independent gene regulation by insulating genes from the effects of enhancers or organized chromatin. However, the mechanisms of boundary action are not well understood. To investigate whether boundary function depends on a higher order of chromatin organization, we examined the function of several Drosophila melanogaster insulators in cells with reduced chromatin-remodeling activities. We found that knockdown of NURF301 and ISWI, key components of the nucleosome-remodeling factor (NURF), synergistically disrupted the enhancer-blocking function of Fab7 and SF1 and augmented the function of Fab8. Mutations in Nurf301/Ebx and Iswi also affected the function of these boundaries in vivo. We further show that ISWI was localized on the endogenous Fab7 and Fab8 insulators and that NURF knockdown resulted in a marked increase in the nucleosome occupancy at these insulator sites. In contrast to the effect of NURF knockdown, reduction in dMi-2, the ATPase component of the Drosophila nucleosome-remodeling and deacetylation (NuRD) complex, augmented Fab7 and suppressed Fab8. Our results provide the first evidence that higher-order chromatin organization influences the enhancer-blocking activity of chromatin boundaries. In particular, the NURF and NuRD nucleosome-remodeling complexes may regulate Hox expression by modulating the function of boundaries in these complexes. The unique responses by different classes of boundaries to changes in the chromatin environment may be indicative of their distinct mechanisms of action, which may influence their placement in the genome and selection during evolution.

scholarly journals A group of scs elements function as domain boundaries in an enhancer-blocking assay.

1992 ◽  
Vol 12 (5) ◽  
pp. 2424-2431 ◽  
Author(s):  
R Kellum ◽  
P Schedl
Keyword(s):  
Fusion Gene ◽  
Boundary Function ◽  
Higher Order ◽  
Gene Activity ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Domain Boundaries ◽  
Enhancer Blocking ◽  
Blocking Assay

Chromosomes of higher eukaryotes are thought to be organized into a series of discrete and topologically independent higher-order domains. In addition to providing a mechanism for chromatin compaction, these higher-order domains are thought to define independent units of gene activity. Implicit in most models for the folding of the chromatin fiber are special nucleoprotein structures, the domain boundaries, which serve to delimit each higher-order chromosomal domain. We have used an "enhancer-blocking assay" to test putative domain boundaries for boundary function in vivo. This assay is based on the notion that in delimiting independent units of gene activity, domain boundaries should be able to restrict the scope of activity of enhancer elements to genes which reside within the same domain. In this case, interposing a boundary between an enhancer and a promoter should block the action of the enhancer. In the experiments reported here, we have used the yolk protein-1 enhancer element and an hsp70 promoter:lacZ fusion gene to test putative boundary DNA segments for enhancer-blocking activity. We have found that several scs-like elements are capable of blocking the action of the yp-1 enhancer when placed between it and the hsp70 promoter. In contrast, a MAR/SAR DNA segment and another spacer DNA segment had no apparent effect on enhancer activity.

A group of scs elements function as domain boundaries in an enhancer-blocking assay

1992 ◽  
Vol 12 (5) ◽  
pp. 2424-2431
Author(s):  
R Kellum ◽  
P Schedl
Keyword(s):  
Fusion Gene ◽  
Boundary Function ◽  
Higher Order ◽  
Gene Activity ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Domain Boundaries ◽  
Enhancer Blocking ◽  
Blocking Assay

Chromosomes of higher eukaryotes are thought to be organized into a series of discrete and topologically independent higher-order domains. In addition to providing a mechanism for chromatin compaction, these higher-order domains are thought to define independent units of gene activity. Implicit in most models for the folding of the chromatin fiber are special nucleoprotein structures, the domain boundaries, which serve to delimit each higher-order chromosomal domain. We have used an "enhancer-blocking assay" to test putative domain boundaries for boundary function in vivo. This assay is based on the notion that in delimiting independent units of gene activity, domain boundaries should be able to restrict the scope of activity of enhancer elements to genes which reside within the same domain. In this case, interposing a boundary between an enhancer and a promoter should block the action of the enhancer. In the experiments reported here, we have used the yolk protein-1 enhancer element and an hsp70 promoter:lacZ fusion gene to test putative boundary DNA segments for enhancer-blocking activity. We have found that several scs-like elements are capable of blocking the action of the yp-1 enhancer when placed between it and the hsp70 promoter. In contrast, a MAR/SAR DNA segment and another spacer DNA segment had no apparent effect on enhancer activity.

scholarly journals Functional Interactions between NURF and Ctcf Regulate Gene Expression

2014 ◽  
Vol 35 (1) ◽  
pp. 224-237 ◽  
Author(s):  
Zhijun Qiu ◽  
Carolyn Song ◽  
Navid Malakouti ◽  
Daniel Murray ◽  
Aymen Hariz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Nucleosome Occupancy ◽  
Cell Types ◽  
Ctcf Binding ◽  
Nucleosome Remodeling ◽  
Functional Interactions ◽  
Chromatin Remodeling Complexes ◽  
Reporter Assays

Gene expression frequently requires chromatin-remodeling complexes, and it is assumed that these complexes have common gene targets across cell types. Contrary to this belief, we show by genome-wide expression profiling that Bptf, an essential and unique subunit of the nucleosome-remodeling factor (NURF), predominantly regulates the expression of a unique set of genes between diverse cell types. Coincident with its functions in gene expression, we observed that Bptf is also important for regulating nucleosome occupancy at nucleosome-free regions (NFRs), many of which are located at sites occupied by the multivalent factors Ctcf and cohesin. NURF function at Ctcf binding sites could be direct, because Bptf occupies Ctcf binding sitesin vivoand has physical interactions with CTCF and the cohesin subunit SA2. Assays of several Ctcf binding sites using reporter assays showed that their regulatory activity requires Bptf in two different cell types. Focused studies atH2-K1showed that Bptf regulates the ability of Klf4 to bind near an upstream Ctcf site, possibly influencing gene expression. In combination, these studies demonstrate that gene expression as regulated by NURF occurs partly through physical and functional interactions with the ubiquitous and multivalent factors Ctcf and cohesin.

scholarly journals Embryonic erythropoiesis and hemoglobin switching require transcriptional repressor ETO2 to modulate chromatin organization

2020 ◽  
Vol 48 (18) ◽  
pp. 10226-10240
Author(s):  
Xiang Guo ◽  
Jennifer Plank-Bazinet ◽  
Ivan Krivega ◽  
Ryan K Dale ◽  
Ann Dean
Keyword(s):  
Target Genes ◽  
Nucleosome Occupancy ◽  
Fetal Liver ◽  
Globin Gene ◽  
Chromatin Organization ◽  
Heptad Repeat ◽  
Enhancer Activity ◽  
Erythroid Progenitor ◽  
Nucleosome Remodeling ◽  
Hemoglobin Switching

Abstract The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human β-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the β-globin locus control region and γ-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal γ-globin/LCR looping and γ-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing γ-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.

scholarly journals PHF20L1 as a H3K27me2 reader coordinates with transcriptional repressors to promote breast tumorigenesis

2020 ◽  
Vol 6 (16) ◽  
pp. eaaz0356 ◽  
Author(s):  
Yongqiang Hou ◽  
Wei Liu ◽  
Xianfu Yi ◽  
Yang Yang ◽  
Dongxue Su ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Transcriptional Repression ◽  
Strongly Correlated ◽  
Nurd Complex ◽  
Breast Tumorigenesis ◽  
Nucleosome Remodeling ◽  
Polycomb Repressive Complex 2 ◽  
Tudor Domain ◽  
Proliferation And Metastasis

TUDOR domain–containing proteins (TDRDs) are chiefly responsible for recognizing methyl-lysine/arginine residue. However, how TDRD dysregulation contributes to breast tumorigenesis is poorly understood. Here, we report that TUDOR domain–containing PHF20L1 as a H3K27me2 reader exerts transcriptional repression by recruiting polycomb repressive complex 2 (PRC2) and Mi-2/nucleosome remodeling and deacetylase (NuRD) complex, linking PRC2-mediated methylation and NuRD-mediated deacetylation of H3K27. Furthermore, PHF20L1 was found to serve as a potential MYC and hypoxia-driven oncogene, promoting glycolysis, proliferation, and metastasis of breast cancer cells by directly inhibiting tumor suppressors such as HIC1, KISS1, and BRCA1. PHF20L1 expression was also strongly correlated with higher histologic grades of breast cancer and markedly up-regulated in several cancers. Meanwhile, Phf20l1 deletion not only induces growth retardation and mammary ductal outgrowth delay but also inhibits tumorigenesis in vivo. Our data indicate that PHF20L1 promotes tumorigenesis, supporting the pursuit of PHF20L1 as a target for cancer therapy.

scholarly journals Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

2018 ◽  
Author(s):  
Leslie Y. Beh ◽  
Galia T. Debelouchina ◽  
Derek M. Clay ◽  
Robert E. Thompson ◽  
Kelsi A. Lindblad ◽  
...  
Keyword(s):  
De Novo ◽  
Nucleosome Occupancy ◽  
Dna Methyltransferases ◽  
Chromatin Organization ◽  
Sexual Cycle ◽  
List Type ◽  
Adenine Methylation ◽  
The Impact

SummaryDNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Here, we report a novel DNA 6mA methyltransferase in ciliates, termed MTA1. The enzyme contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1in vivoleads to the genome-wide loss of 6mA in asexually growing cells and abolishment of the consensus ApT dimethylated motif. Genes exhibit subtle changes in chromatin organization or RNA expression upon loss of 6mA, depending on their starting methylation level. Mutants fail to complete the sexual cycle, which normally coincides with a peak of MTA1 expression. Thus, MTA1 functions in a developmental stage-specific manner. We determine the impact of 6mA on chromatin organizationin vitroby reconstructing complete, full-length ciliate chromosomes harboring 6mA in native or ectopic positions. Using these synthetic chromosomes, we show that 6mA directly disfavors nucleosomesin vitroin a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a novel MT-A70 protein necessary for eukaryotic 6mA methylation and defines the impact of 6mA on chromatin organization using epigenetically defined synthetic chromosomes.HighlightsThe MT-A70 protein MTA1 mediates DNA N6-adenine methylation inOxytrichaMTA1 mutants exhibit subtle changes in nucleosome organization and transcriptionin vivo6mA directly disfavors nucleosome occupancy in natural and synthetic chromosomesin vitroDe novosynthesis of complete, epigenetically definedOxytrichachromosomes

scholarly journals The Role of Non-Catalytic Domains of Hrp3 in Nucleosome Remodeling

2021 ◽  
Vol 22 (4) ◽  
pp. 1793
Author(s):  
Wenbo Dong ◽  
Punit Prasad ◽  
Andreas Lennartsson ◽  
Karl Ekwall
Keyword(s):  
Chromatin Remodeling ◽  
Nucleosome Occupancy ◽  
Strong Association ◽  
Atpase Domain ◽  
Coupling Region ◽  
Nucleosome Remodeling ◽  
Catalytic Domains

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.

scholarly journals Dinucleosome specificity and allosteric switch of the ISW1a ATP-dependent chromatin remodeler in transcription regulation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Saurabh K. Bhardwaj ◽  
Solomon G. Hailu ◽  
Lola Olufemi ◽  
Sandipan Brahma ◽  
Soumyadipta Kundu ◽  
...  
Keyword(s):  
Transcription Regulation ◽  
Histone Acetyltransferase ◽  
Mutational Analysis ◽  
Higher Order ◽  
Chromatin Organization ◽  
Atpase Domain ◽  
Nucleosome Remodeling ◽  
Chromatin Remodelers ◽  
Nucleosomal Dna ◽  
Accessory Subunit

AbstractOver the last 3 decades ATP-dependent chromatin remodelers have been thought to recognize chromatin at the level of single nucleosomes rather than higher-order organization of more than one nucleosome. We show the yeast ISW1a remodeler has such higher-order structural specificity, as manifested by large allosteric changes that activate the nucleosome remodeling and spacing activities of ISW1a when bound to dinucleosomes. Although the ATPase domain of Isw1 docks at the SHL2 position when ISW1a is bound to either mono- or di-nucleosomes, there are major differences in the interactions of the catalytic subunit Isw1 with the acidic pocket of nucleosomes and the accessory subunit Ioc3 with nucleosomal DNA. By mutational analysis and uncoupling of ISW1a’s dinucleosome specificity, we find that dinucleosome recognition is required by ISW1a for proper chromatin organization at promoters; as well as transcription regulation in combination with the histone acetyltransferase NuA4 and histone H2A.Z exchanger SWR1.

scholarly journals Splice Variants of the SWR1-type Nucleosome Remodeling Factor Domino Have Distinct Functions during Drosophila melanogaster Oogenesis

2016 ◽  
Author(s):  
Kenneth Boerner ◽  
Peter B. Becker
Keyword(s):  
Drosophila Melanogaster ◽  
Splice Variants ◽  
Female Sterility ◽  
Nurse Cells ◽  
Histone H2a ◽  
Cell Type ◽  
Nucleosome Remodeling ◽  
Cellular Processes ◽  
Cell Type Specific

AbstractSWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed, but not well documented in vivo. It is also unclear, whether the two alternatively spliced isoforms, dom-A and dom-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis causing female sterility. Therefore, we systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg chamber formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, while incorporation in endoreplicating germline nurse cells is independent of DOM. In contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange.Summary StatementIsoforms of nucleosome remodeling factor Domino change chromatin structure by histone variant exchange to direct essential cellular processes in oocyte development.

Insights into amyloid disease from fly models

2014 ◽  
Vol 56 ◽  
pp. 69-83 ◽  
Author(s):  
Ko-Fan Chen ◽  
Damian C. Crowther
Keyword(s):  
Drosophila Melanogaster ◽  
Neurodegenerative Disorders ◽  
Amyloid Β ◽  
Amyloid Β Peptide ◽  
Disease Pathogenesis ◽  
Amyloid Aggregates ◽  
Aβ Amyloid ◽  
Polypeptide Chains ◽  
Amyloid Diseases

The formation of amyloid aggregates is a feature of most, if not all, polypeptide chains. In vivo modelling of this process has been undertaken in the fruitfly Drosophila melanogaster with remarkable success. Models of both neurological and systemic amyloid diseases have been generated and have informed our understanding of disease pathogenesis in two main ways. First, the toxic amyloid species have been at least partially characterized, for example in the case of the Aβ (amyloid β-peptide) associated with Alzheimer's disease. Secondly, the genetic underpinning of model disease-linked phenotypes has been characterized for a number of neurodegenerative disorders. The current challenge is to integrate our understanding of disease-linked processes in the fly with our growing knowledge of human disease, for the benefit of patients.

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