scholarly journals Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1

2020 ◽  
Vol 48 (14) ◽  
pp. 7728-7747 ◽  
Author(s):  
Maria Tauber ◽  
Sarah Kreuz ◽  
Alexander Lemak ◽  
Papita Mandal ◽  
Zhadyra Yerkesh ◽  
...  
Keyword(s):  
Histone Modification ◽  
Cellular Localization ◽  
Divergent Evolution ◽  
Chromatin Binding ◽  
Linker Region ◽  
Binding Domains ◽  
Splicing Variants ◽  
Epigenetic Regulator ◽  
Histone Ubiquitylation ◽  
Binding Behaviour

Abstract UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.

Identification of two independent nucleosome-binding domains in the transcriptional co-activator SPBP

2012 ◽  
Vol 442 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Sagar Darvekar ◽  
Sylvia Sagen Johnsen ◽  
Agnete Bratsberg Eriksen ◽  
Terje Johansen ◽  
Eva Sjøttem
Keyword(s):  
Transcription Factors ◽  
Dependent Manner ◽  
Chromatin Binding ◽  
Binding Region ◽  
Interaction Domain ◽  
Binding Domains ◽  
Element Binding Protein ◽  
Inducible Protein ◽  
Responsive Element ◽  
Nucleosome Binding

Transcriptional regulation requires co-ordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. In the present study we demonstrate that the transcriptional co-regulator SPBP [stromelysin-1 PDGF (platelet-derived growth factor)-responsive element binding protein] contains two independent chromatin-binding domains, the SPBP-(1551–1666) region and the C-terminal extended PHD [ePHD/ADD (extended plant homeodomain/ATRX-DNMT3-DNMT3L)] domain. The region 1551–1666 is a novel core nucleosome-interaction domain located adjacent to the AT-hook motif in the DNA-binding domain. This novel nucleosome-binding region is critically important for proper localization of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail-dependent manner, and has significant impact on the dynamic interaction between SPBP and chromatin. Furthermore, SPBP and its homologue RAI1 (retinoic-acid-inducible protein 1), are strongly enriched on chromatin in interphase HeLa cells, and both proteins display low nuclear mobility. RAI1 contains a region with homology to the novel nucleosome-binding region SPBP-(1551–1666) and an ePHD/ADD domain with ability to bind nucleosomes. These results indicate that the transcriptional co-regulator SPBP and its homologue RAI1 implicated in Smith–Magenis syndrome and Potocki–Lupski syndrome both belong to the expanding family of chromatin-binding proteins containing several domains involved in specific chromatin interactions.

Molecular investigation of the TTD and PHD histone binding domains of the epigenetic regulator UHRF2 .

2021 ◽  
Author(s):  
Shane M. Ginnard ◽  
Alyssa E. Winkler ◽  
Carlos Mellado Fritz ◽  
Tatum Bluhm ◽  
Ray Kemmer ◽  
...  
Keyword(s):  
Histone Binding ◽  
Binding Domains ◽  
Epigenetic Regulator ◽  
Molecular Investigation

scholarly journals Two Copies of mthmg1, Encoding a Novel Mitochondrial HMG-Like Protein, Delay Accumulation of Mitochondrial DNA Deletions in Podospora anserina

2002 ◽  
Vol 1 (4) ◽  
pp. 503-513 ◽  
Author(s):  
Michelle Dequard-Chablat ◽  
Cynthia Alland
Keyword(s):  
Mitochondrial Dna ◽  
Dna Binding ◽  
Mitochondrial Genome ◽  
Life Span ◽  
Cellular Localization ◽  
Premature Death ◽  
Podospora Anserina ◽  
Protein Fusion ◽  
Dna Binding Domains ◽  
Binding Domains

ABSTRACT In the filamentous fungus Podospora anserina, two degenerative processes which result in growth arrest are associated with mitochondrial genome (mitochondrial DNA [mtDNA]) instability. Senescence is correlated with mtDNA rearrangements and amplification of specific regions (senDNAs). Premature death syndrome is characterized by the accumulation of specific mtDNA deletions. This accumulation is due to indirect effects of the AS1-4 mutation, which alters a cytosolic ribosomal protein gene. The mthmg1 gene has been identified as a double-copy suppressor of premature death. It greatly delays premature death and the accumulation of deletions when it is present in two copies in an AS1-4 context. The duplication of mthmg1 has no significant effect on the wild-type life span or on senDNA patterns. In an AS1 + context, deletion of the mthmg1 gene alters germination, growth, and fertility and reduces the life span. The Δmthmg1 senescent strains display a particular senDNA pattern. This deletion is lethal in an AS1-4 context. According to its physical properties (very basic protein with putative mitochondrial targeting sequence and HMG-type DNA-binding domains) and the cellular localization of an mtHMG1-green fluorescent protein fusion, mtHMG1 appears to be a mitochondrial protein possibly associated with mtDNA. It is noteworthy that it is the first example of a protein combining the two DNA-binding domains, AT-hook motif and HMG-1 boxes. It may be involved in the stability and/or transmission of the mitochondrial genome. To date, no structural homologues have been found in other organisms. However, mtHMG1 displays functional similarities with the Saccharomyces cerevisiae mitochondrial HMG-box protein Abf2.

The Nucleophosmin-1 Splice Variant Analysis Provides More Important Information On Prognosis Than NPM1 Mutational Status In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2563-2563
Author(s):  
Krzysztof Giannopoulos ◽  
Malgorzata Zajac ◽  
Anna Dolnik ◽  
Stephany Correa ◽  
Konstanze Dohner ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Variant ◽  
Research Funding ◽  
Cellular Localization ◽  
Prognostic Significance ◽  
Splicing Variant ◽  
Mutational Status ◽  
Splicing Variants ◽  
Survival Differences ◽  
Median Expression

Abstract Background The process of mRNA splicing has been reported to play an important role in human disease development and many cancer-related genes are regulated by alternative splicing. In addition, first analyses of alternative splicing in bone marrow of AML samples identified novel splice variants specific for AML patients in comparison to normal cells such NOTCH2, CD13 and FLT3. Recently, NPM1 mutations have been included as novel provisional entity within the WHO classification of AML. This new entity bears distinct genetic, pathological and clinical features. Of particular importance is the fact that mutations in NPM1 without concomitant FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations identifies a group of cytogenetically normal (CN) AML patients with favorable prognosis. Since splicing variants play an important role in cellular functioning and splicing factor mutations have been reported in myeloid tumors including AML, the current study focuses on the characterization of NPM1splicing variants expression as well as its impact on the biology and prognosis of AML patients. Methods For 104 samples (52 CN-AML and 52 patients samples with cytogenetic aberrations) qRT-PCR was performed. For sensitivity data normalization β-actin (ACTB)was analyzed. Quantity mean values for gene expression were calculated according to the Standard Curve method. In this first cohort of patients total expression of NPM1 (Rt) as well as levels of the three splicing variants of NPM1 were evaluated: R1 that translates exon 1 to 9 and 11 to 12, R2 which contains exons 1 to 10 and R3 that lacks exons 8 and 10. We found prognostic significance of the expression level of NPM1-R2, therefore we decided to validate the prognostic significance of the expression of NPM1-R2 in independent cohort of AML patients. We consolidated 104 patients previously analyzed with 87 patients from the new cohort and preformed the final analysis for NPM1-R2 in total 191 cases. The existence of NPM1-R2 at the protein level was evaluated with the use of Western Blot technique. The cellular localization of NPM-1 was assessed by immunohistochemistry and analyzed with the respect to NPM1-R2 expression. Results Total expression as well as expression of splicing variants R1 and R3 were significantly higher in 104 AML patients compared to healthy volunteers (HVs)with a median expression of 8.587 vs 0.928 (p= 0.001), 1.729 vs 0.5485 (p=0.014), and 2.535 vs 0.108 (p<0.0001), respectively. We evaluated the existence of NPM1-R2 at the protein in AML samples as well as AML cell line KG1. We found that the expression of R2 splicing variant was significantly higher in all AML patients compared to HVs with a median expression of 1.64 vs 0.33 (p= 0.009, n=191)). We have found no differences between groups of AML patients with and without NPM1 mutations (1.21 vs 0.82, p= 0.13). High R2 splicing variant expression was associated with longer OS when CN-AML patients were analyzed (880 vs 438 days, p= 0.028), but there was no association with OS in case of high or low R2 expression in all AML patients. Longer OS was observed in CN-AML patients with high R2 expression without concomitant FLT3-ITD mutations compared to the rest of groups (p<0.0001). Most importantly, in our cohort of CN-AML cases survival differences seen between the established ELN groups according to a NPM1/FLT3-ITD stratification were less impressive than between groups stratified according to R2 expression combined with FLT3-ITD mutational status. Conclusion Since the R2 splicing variant represents a truncated form of NPM1 gene due to the of the lack of exons 11 and 12 (coding for the domain responsible for nucleolar localization of the protein), this isoform mostly localizes in the nucleoplasm, and thus might also have a biological impact in the malignant cells. Most importantly, in our cohort of cases survival differences seen between the established ELN groups according to a NPM1/FLT3-ITD stratification were less impressive than between groups stratified according to R2 expression combined with FLT3-ITD mutational status. In summary, the expression of NPM1-R2 might be of biological importance for CN-AML patients. Moreover, R2 splice variant provides prognostic value for CN-AML patients and should be assessed in addition the NPM1 mutational status. Disclosures: Schlenk: Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Chugai: Research Funding; Ambit: Honoraria.

Inventory and analysis of ATP-binding cassette (ABC) systems inBrugia malayi

Parasitology ◽  
2010 ◽  
Vol 137 (8) ◽  
pp. 1195-1212 ◽  
Author(s):  
B. F. ARDELLI ◽  
L. E. STITT ◽  
J. B. TOMPKINS
Keyword(s):  
Drug Resistance ◽  
Dna Repair ◽  
Abc Transporters ◽  
Atp Binding ◽  
Switch Region ◽  
Linker Region ◽  
Protein Superfamilies ◽  
Binding Domains ◽  
Reverse Transcription Pcr ◽  
Signature Sequence

SUMMARYABC systems are one of the largest described protein superfamilies. These systems have a domain organization that may contain 1 or more transmembrane domains (ABC_TM1F) and 1 or 2 ATP-binding domains (ABC_2). The functions (e.g., import, export and DNA repair) of these proteins distinguish the 3 classes of ABC systems. Mining and PCR-based cloning were used to identify 33 putative ABC systems from theBrugia malayigenome. There were 31 class 2 genes, commonly called ABC transporters, and 2 class 3 genes. The ABC transporters were divided into subfamilies. Three belonged to subfamily A, 16 to subfamily B, 5 to subfamily C, 1 to subfamily E and 3 to subfamilies F and G, respectively. None were placed in subfamilies D and H. Similar to other ABC systems, the ABC_2 domain ofB. malayigenes was conserved and contained the Walker A and B motifs, the signature sequence/linker region and the switch region with the conserved histidine. The ABC_TM1F domain was less conserved. The relative abundance of ABC systems was quantified using real-time reverse transcription PCR and was significantly higher in female adults ofB. malayithan in males and microfilaria, particularly those in subfamilies B and C, which are associated with drug resistance.

Isoform-dependent subcellular localization of LMTK1A and LMTK1B and their roles in axon outgrowth and spine formation

2020 ◽  
Vol 168 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Ran Wei ◽  
Arika Sugiyama ◽  
Yuta Sato ◽  
Motohiro Nozumi ◽  
Hironori Nishino ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Integral Membrane Protein ◽  
Axon Outgrowth ◽  
Endosomal Trafficking ◽  
Binding Modes ◽  
Wild Type ◽  
Spine Formation ◽  
Splicing Variants ◽  
Membrane Bound ◽  
Tubular Form

Abstract Lemur kinase 1 (LMTK1) is a membrane-bound Ser/Thr kinase that is expressed in neurons. There are two splicing variants of LMTK1 with different membrane binding modes, viz., cytosolic LMTK1A that binds to membranes through palmitoylation at the N-terminal cysteines and LMTK1B, an integral membrane protein with transmembrane sequences. We recently reported that LMTK1A regulates axon outgrowth and spine formation in neurons. However, data about LMTK1B are scarce. We analysed the expression and cellular localization of LMTK1B along with its role in axon and spine formation. We found that both LMTK1B and LMTK1A were expressed equally in the cerebral cortex and cerebellum of the mouse brain. Similar to LMTK1A, the wild type of LMTK1B was localized to Rab11-positive pericentrosomal compartment. The kinase negative (kn) mutant of LMTK1B was found to be associated with an increase in the tubular form of endoplasmic reticulum (ER), which was not the case with LMTK1A kn. Furthermore, unlike LMTK1A kn, LMTK1B kn did not stimulate the axon outgrowth and spine formation. These results suggest that while LMTK1A and LMTK1B share a common function in recycling endosomal trafficking at the pericentrosomal compartment, LMTK1B has an additional unique function in vesicle transport in the ER region.

scholarly journals Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

2019 ◽  
Author(s):  
Andrew R Harris ◽  
Brian Belardi ◽  
Pamela Jreij ◽  
Kathy Wei ◽  
Hengameh Shams ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Negative Regulator ◽  
Actin Binding ◽  
Binding Properties ◽  
High Sequence Similarity ◽  
Calponin Homology ◽  
Binding Domains

ABSTRACTTandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for f-actin. To investigate features that affect CH1-CH2 affinity for f-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a PEG polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective ‘openness’ of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution ‘openness’, suggesting additional influences on affinity. Interestingly, we also observe non-uniform sub-cellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties.

scholarly journals Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications

2002 ◽  
Vol 366 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Martina GENTZSCH ◽  
Andrei ALEKSANDROV ◽  
Luba ALEKSANDROV ◽  
John R. RIORDAN
Keyword(s):  
Cystic Fibrosis ◽  
Cellular Localization ◽  
Three Dimensional ◽  
Large Family ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Abc Proteins ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163–180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final β-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413–1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291–1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate β-strand of the domain which is likely to participate in essential structure-stabilizing β-sheet formation.

scholarly journals The topology of chromatin-binding domains in the NuRD deacetylase complex

2020 ◽  
Vol 48 (22) ◽  
pp. 12972-12982
Author(s):  
Christopher J Millard ◽  
Louise Fairall ◽  
Timothy J Ragan ◽  
Christos G Savva ◽  
John W R Schwabe
Keyword(s):  
Electron Microscopy ◽  
Associated Factors ◽  
Chromatin Binding ◽  
Potential Mechanism ◽  
Nurd Complex ◽  
Core Complex ◽  
The Core ◽  
Binding Domains ◽  
Cryo Electron Microscopy ◽  
Nuclear Processes

Abstract Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.

Sign in / Sign up

Export Citation Format

Share Document