scholarly journals Characterization and evolutionary origin of novel C2H2 zinc finger protein (ZNF648) required for both erythroid and megakaryocyte differentiation in humans

Haematologica ◽  
2020 ◽  
pp. 0-0
Author(s):  
Daniel C. J. Ferguson ◽  
Juraidah Haji Mokim ◽  
Marjolein Meinders ◽  
Edmund R. R. Moody ◽  
Tom A. Williams ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Sequence Similarity ◽  
Zinc Finger Domain ◽  
Erythroid Cells ◽  
Coding Region ◽  
Finger Protein ◽  
C Terminus ◽  
N Terminus ◽  
Megakaryocyte Differentiation

Human ZNF648 is a novel poly C-terminal C2H2 zinc finger protein identified amongst the most dysregulated proteins in erythroid cells differentiated from iPSC. Its nuclear localisation and structure indicate it is likely a DNA-binding protein. Using a combination of ZNF648 overexpression in an iPSC line and primary adult erythroid cells, ZNF648 knockdown in primary adult erythroid cells and megakaryocytes, comparative proteomics and transcriptomics we show that ZNF648 is required for both erythroid and megakaryocyte differentiation. Orthologues of ZNF648 were detected across Mammals, Reptilia, Actinopterygii, in some Aves, Amphibia and Coelacanthiformes suggesting the gene originated in the common ancestor of Osteichthyes (Euteleostomi or bony fish). Conservation of the C-terminal zinc finger domain is higher, with some variation in zinc finger number but a core of at least six zinc fingers conserved across all groups, with the N-terminus recognisably similar within but not between major lineages. This suggests the N-terminus of ZNF648 evolves faster than the C-terminus, however this is not due to exon-shuffling as the entire coding region of ZNF648 is within a single exon. As for other such transcription factors, the N-terminus likely carries out regulatory functions, but showed no sequence similarity to any known domains. The greater functional constraint on the zinc finger domain suggests ZNF648 binds at least some similar regions of DNA in the different organisms. However, divergence of the N-terminal region may enable differential expression, allowing adaptation of function in the different organisms.

CoCoA/CCAR1 Pair-Mediated Recruitment of Mediator Complex Indicates Novel Pathway for the Function of GATA1 in Erythroid Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1302-1302
Author(s):  
Chihiro Kaminaga ◽  
Shumpei Mizuta ◽  
Tomoya Minami ◽  
Kasumi Oda ◽  
Haruka Fujita ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Direct Interaction ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Zinc Finger Domain ◽  
C Terminus ◽  
N Terminus ◽  
Reporter Assays ◽  
Globin Promoter

Abstract Abstract 1302 The mammalian multi-protein complex Mediator, originally identified by ourselves as a nuclear receptor-specific coactivator complex, is a phylogenetically-conserved subcomplex of the RNA polymerase II holoenzyme and serves as an end-point integrator of diverse intracellular signals and transcriptional activators. The 220-kDa Mediator subunit MED1 is a specific coactivator not only for nuclear receptors but for GATA family activators, and serves as a GATA1-specific coactivator that is essential for optimal GATA1-mediated erythropoiesis. In this study, we show a novel nuclear signaling pathway for MED1 action in GATA1-induced transcriptional activation during erythroid differentiation. First, we identified the amino acid residues 681–715 of human MED1 (MED1(aa.681-715)) to be responsible for the direct interaction with GATA1. When MED1 in K562 human erythroleukemic cells was knocked down during hemin-induced erythroid differentiation, the erythroid differentiation was significantly attenuated as assessed by an erythroid differentiation score defined by the number of cells positive for benzidine staining, and the expressions of the GATA1-targeted and erythroid differentiation marker genes, β-globin, γ-globin, PBGD and ALAS-E, were prominently attenuated. However, overexpressions of the N-terminal MED1 truncations without and with nuclear receptor recognition motifs, MED1(aa.1–602) and MED1(aa.1–703), respectively, but neither of which could bind to GATA1 (above), prominently enhanced erythroid differentiation of K562 cells. Luciferase reporter assays by using the human γ-globin promoter and Med1−/− mouse embryonic fibroblasts (MEFs) showed that these N-terminal MED1 truncations rescued GATA1-mediated transactivation, indicating that MED1(a.a.1–602) served as the functional interaction surface for GATA1. Hence, a putative bypass for GATA1-MED1 pathway appears to exist, and is expected to interact with the N-terminus of MED1. As a candidate bypass system, we tested both the recently reported bypass molecule for a nuclear post-activator signaling, CCAR1, and its partner coactivator CoCoA. CCAR1 was reported by others to bypass the estrogen receptor-mediated transactivation by a simultaneous binding of CCAR1 with the estrogen receptor and the N-terminus of MED1. Functionally, serial luciferase reporter assays by using the γ-globin promoter and MEFs demonstrated cooperative transactivation by combinations of GATA1, CCAR1, CoCoA and/or the N-terminus of MED1, but the transactivation mediated by the N-terminus of MED1 was not as prominent as the one mediated by the full-length MED1. An overexperssion of CCAR1 or CoCoA in K562 cells prominently enhanced both the GATA1-mediated erythroid differentiation and the expressions of the GATA1-targeted genes. Next, the mechanisms underlying the CCAR1- and CoCoA-mediated GATA1 functions were analyzed by serial GST-pulldown and mammalian two-hybrid assays, and the following results were obtained. (i) The N-terminus of CCAR1 interacted with the C-terminus of CoCoA. (ii) The N-terminus of MED1 interacted with both the N- and C-termini of CCAR1. (iii) While the N-terminal zinc-finger domain of human GATA1 (GATA1(a.a.204–228)) is known to bind to the well-known GATA1 partner FOG1, intriguingly, the C-terminal zinc-finger domain of GATA1 (GATA1(a.a.258–272)) interacted with all three of the following cofactors; MED1 (MED1(aa.681–715)), CCAR1 (at the C-terminus) and CoCoA (at both the N- and C-termini). The affinity of CoCoA to bind to GATA1 appeared to be a little higher than the other. Thus, the GATA1(a.a.258-272) zinc finger appears to serve as a docking surface for multiple coactivating proteins, where both MED1 and CoCoA/CCAR1 pair can interact, probably in a competitive manner, or perhaps simultaneously. Here, both CoCoA/CCAR1 as a pair and CCAR1 by itself can serve as a bypass. Finally, ChIP assays of hemin-treated K562 cells showed that GATA1, CCAR1/CoCoA and MED1 were all recruited onto the γ-globin promoter during transactivation. Taken together, besides a direct interaction between GATA1 and MED1, the CoCoA/CCAR1 pair appears to relay the GATA1 signal to MED1. The multiple modes of mechanisms for transcription mediated by the GATA1-MED1 axis might contribute to a fine tuning of the GATA1 function, not only during erythropoiesis but also in other GATA1-mediated homeostasis events, within a living animal. Disclosures: No relevant conflicts of interest to declare.

The artificial 4-zinc-finger protein Bagly binds human utrophin promoter A at the endogenous chromosomal site and activates transcription

2007 ◽  
Vol 85 (3) ◽  
pp. 358-365 ◽  
Author(s):  
Annalisa Onori ◽  
Agata Desantis ◽  
Serena Buontempo ◽  
Maria Grazia Di Certo ◽  
Maurizio Fanciulli ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Transcriptional Activity ◽  
Zinc Finger Protein ◽  
Target Sequence ◽  
Zinc Finger Domain ◽  
Active Chromatin ◽  
Finger Protein ◽  
Chromosomal Site ◽  
Transcription Factor Yy1 ◽  
Test Gene

Our aim is to upregulate the expression of the dystrophin-related gene utrophin in Duchenne muscular dystrophy, in this way complementing the lack of dystrophin function. To achieve utrophin upregulation, we designed and engineered synthetic zinc-inger based transcription factors. We have previously shown that the artificial 3-zinc-finger protein Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from utrophin promoter A. Here we report a novel artificial 4-zinc-finger protein, Bagly, which binds with optimized affinity–specificity to a 12 bp DNA target sequence that is internal to human utrophin promoter A. Bagly was generated adding to Jazz protein an extra-fourth zinc finger, derived from transcription factor YY1. Importantly, the Bagly DNA target sequence is statistically present in the human genome only 210 times, about 60 fewer times than the 9 bp Jazz DNA target sequence. Thanks to its additional zinc-finger domain, Bagly protein shows enhanced transcriptional activity. Moreover, we demonstrated Bagly's effective access and binding to active chromatin in the chromosomal context and its ability to upregulate endogenous utrophin.

scholarly journals ZAP, a CCCH-Type Zinc Finger Protein, Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication and Interacts with Viral Nsp9

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
Yongxiang Zhao ◽  
Zhongbao Song ◽  
Juan Bai ◽  
Xuewei Liu ◽  
Hans Nauwynck ◽  
...  
Keyword(s):  
Amino Acids ◽  
Zinc Finger ◽  
Transcriptome Sequencing ◽  
Zinc Finger Protein ◽  
Early Stage ◽  
Respiratory Syndrome Virus ◽  
Type I ◽  
Zinc Finger Domain ◽  
Antiviral Protein ◽  
Finger Protein

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens affecting many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. PRRSV infection can cleave mitochondrial antiviral signaling protein (MAVS) and inhibit the induction of type I interferon. The antiviral effector molecules that are involved in host protective responses to PRRSV infection are not fully understood. Here, by using transcriptome sequencing, we found that a zinc finger antiviral protein, ZAP, is upregulated in MAVS-transfected Marc-145 cells and that ZAP suppresses PRRSV infection at the early stage of replication. We also found that the viral protein Nsp9, an RNA-dependent RNA polymerase (RdRp), interacts with ZAP. The interacting locations were mapped to the zinc finger domain of ZAP and N-terminal amino acids 150 to 160 of Nsp9. These findings suggest that ZAP is an effective antiviral factor for suppressing PRRSV infection, and they shed light on virus-host interaction.IMPORTANCEPRRSV continues to adversely impact the global swine industry. It is important to understand the various antiviral factors against PRRSV infection. Here, a zinc finger protein, termed ZAP, was screened from MAVS-induced antiviral genes by transcriptome sequencing, and it was found to remarkably suppress PRRSV replication and interact with PRRSV Nsp9. The zinc finger domain of ZAP and amino acids 150 to 160 of Nsp9 are responsible for the interaction. These findings expand the antiviral spectrum of ZAP and provide a better understanding of ZAP antiviral mechanisms, as well as virus-host interactions.

Burkitt Lymphoma Specific Zinc Finger Protein ZNF385B Is Involved in Regulation of B Cell Apoptosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1584-1584
Author(s):  
Kazutoshi Iijima ◽  
Hiroyuki Yamada ◽  
Masashi Miharu ◽  
Atsuko Nakazawa ◽  
Junichiro Fujimoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Zinc Finger ◽  
Expression Profiling ◽  
Zinc Finger Protein ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Finger Protein ◽  
N Terminus

Abstract Abstract 1584 Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are common types of childhood mature B-cell non-Hodgkin's lymphoma (NHL). Both are postulated to be derived from GC B-cells but they are independent disease entities of NHL; however, they have overlapping morphologic and immunophenotypic features. A characteristic translocation t(8;14) of BL also occurs in 5 to 10 percent of cases of DLBCL. It is not uncommon to have difficulty in differentiating BL from DLBCL at initial diagnosis. Therefore, the latest WHO classification of tumors of the lymphoid tissue has recognized a category with overlapping features between BL and DLBCL. Considering this background, gene-expression profiling using microarray should provide an accurate, quantitative method for distinguishing the NHL. Actually, other researchers performed gene-expression profiling of mature B-cell lymphoma at all ages and showed that BL could be reliably distinguished from DLBCL. It has also been reported that DLBCL can be classified into two categories of patients with very different five-year overall survival rates on the basis of gene-expression profile. In an attempt to investigate the molecules specifically expressed in each type of NHL, we performed the screening of genes expressed characteristically in each type of childhood hematologic malignancy by employing a microarray system using clinical specimens from pediatric patients, consisting of 3 patients with DLBCL, 16 with BL, 6 with BCP-ALL and 6 with T-ALL. Consistent with previous reports, CD40, EBI3, FGD6, LMO2, and SERPINA9 were over-expressed in DLBCL. In addition, over-expression of IL21R, STAT3, BCL6, CD58, JAK3 was observed in DLBCL. In BL, on the other hand, BMP7 and SOX11 were over-expressed as reported previously. Furthermore, we found that ZNF385B, BMP3, PEG10, MUC4, AICDA, SMAD1, C13orf15, CD24 were over-expressed in BL. In these candidate genes, ZNF385B, also called ZNF533, was expressed in vast majority of BL cases as well as BL cell lines but not in other hematopoietic malignancies, including DLBCL, B-cell precursor ALL, and mature B-cell malignancies originate from another developmental stages of B cells. Therefore, we concluded that ZNF385B is a BL-specific zinc-finger protein and can be used as a diagnostic marker for BL. Currently three isoforms (IFs) of ZNF385B have been identified and IF-1 is the longest transcript variant, which possesses 4 ZF domains, while IF-2/3 are shorter transcript variants with 3 ZF domains. Although the function and biological significance of this protein have not been clarified at all, ZNF385A has high homology with ZNF385B IF-2/3 is known to be involved in apoptosis regulation. Therefore, we intended to elucidate the functional role of ZNF385B in B cells. First we analyzed intracellular localization of ZNF385B. When ZNF385B IF-1 proteins fused with EGFP at the N-terminus were expressed transiently in DLBCL BJAB cells, we observed the nuclear localization by obtaining fluorescent images. Next, to examine the characteristics of ZNF385B, we established a protein inducible system in a tetracycline-dependent manner for both ZNF385B IF-1 and its deletion mutant ZNF385B-DEL corresponds to IF-2/3 lacking ZF domain in N-terminus in BJAB cells. Interestingly, ectopic expression of ZNF385B IF-1 induced up-regulation of PERP (p53 apoptosis effector related to PMP-22) and activation of caspase-3 and −8, resulting in apoptosis induction, whereas ZNF385B-DEL did not. Furthermore, ZNF385B-DEL inhibited apoptosis induced by CD20 cross-linking and BCR stimulation. Immunoprecipitation by anti-p53 antibody indicated the binding of ZNF385B isoform 1 with p53. Since PERP is known to be a p53 transcriptional target, these results suggest the involvement of ZNF385B in B-cell apoptosis by modulating p53 transactivation. Considering our observation of ZNF385B expression in a portion of cells in peripheral lymphoid organs, ZNF385B possibly is involved in the regulation of death and survival that specifically occurs in germinal center B cells. Disclosures: No relevant conflicts of interest to declare.

Using Forced Chromatin Looping To Overcome Developmental Silencing Of Embryonic and Fetal β-Type Globin Genes In Adult Erythroid Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 433-433
Author(s):  
Jeremy W Rupon ◽  
Wulan Deng ◽  
Hongxin Wang ◽  
Philip D Gregory ◽  
Andreas Reik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Long Range ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Fetal Liver ◽  
Globin Gene ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Chromatin Looping ◽  
Finger Protein

Abstract The β-genes undergo developmental activation and silencing in part by competing for their upstream enhancer, the locus control region (LCR). In adult erythroid cells, the LCR contacts the β-globin gene promoter by forming a loop that precludes interaction with the embryonic and fetal β-type globin genes. Reversing this developmental gene expression switch in favor of embryonic/fetal genes has therapeutic implications for patients with hemoglobinopathies. Here we employed a forced chromatin looping approach to activate the silenced murine embryonic βh1-globin gene and the human fetal γ-globin gene in adult erythroid cells. We have previously shown that forced recruitment via artificial zinc finger proteins of Ldb1, a protein necessary for long-range chromatin interactions at the β-globin locus, can trigger chromatin loop formation and transcription initiation. Here, we designed a zinc finger protein targeting the βh1 promoter, fused it to the self-association domain of Ldb1 (βh1-Ldb1), and introduced it into an adult murine erythroid cell line that normally produces nearly 100% adult β-globin. βh1-Ldb1 expression activated βh1-globin transcription up to 3000-fold accounting for ∼20% of total β-globin expression. βh1-Ldb1 similarly increased expression of βh1-globin in fetal liver derived primary erythroid cells. These results are striking given the degree to which murine embryonic globin genes are normally repressed. To test whether the activity βh1-Ldb1 was due to a looped interaction of the βh1 promoter with the LCR, we introduced βh1-Ldb1 into fetal liver derived erythroblasts from mice in which the LCR had been deleted. βh1-Ldb1 was virtually inactive in the absence of the LCR, demonstrating the dependence on the LCR and, by inference, long range looping of βh1-Ldb1 function. We next extended this approach to the human β-globin locus in an effort to activate expression of the fetal γ-globin gene in adult erythroid cells. Ldb1 was fused to a previously described γ-globin promoter binding zinc finger protein, GG1, to generate GG1-Ldb1. Introduction of GG1-Ldb1 into adult primary human erythroid cells strongly activated γ-globin expression with a concomitant reduction in β-globin transcription. Strikingly, γ-globin accounted for nearly 90% of total β-type globin transcription. Furthermore, fetal hemoglobin expression was nearly pan-cellular as determined by flow cytometry. These results demonstrate the power of forced chromatin looping to reprogram developmental regulation of gene expression, and provide a novel proof of concept for activating the γ-globin gene for the benefit of patients with hemoglobinopathies. Disclosures: Gregory: Sangamo BioSciences: Employment. Reik:Sangamo BioSciences: Employment.

scholarly journals The zinc-finger protein Red1 orchestrates MTREC submodules and binds the Mtl1 helicase arch domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nikolay Dobrev ◽  
Yasar Luqman Ahmed ◽  
Anusree Sivadas ◽  
Komal Soni ◽  
Tamás Fischer ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Mutational Analysis ◽  
Zinc Finger Domain ◽  
Yeast Two Hybrid ◽  
Finger Protein ◽  
Nuclear Exosome ◽  
Interaction Map ◽  
Two Hybrid

AbstractCryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.

Sign in / Sign up

Export Citation Format

Share Document