Plant Homeodomain Zinc Finger Research Articles

Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach.

Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers.

1H, 13C and 15N resonance assignments and secondary structure of the human PHF6-ePHD1 domain.

The plant homeodomain (PHD) finger 6 (PHF6) is a multidomain protein that comprises four nuclear localization signals and two extended PHD zinc finger domains (ePHD), suggesting that the PHD domains of PHF6 may have different functions compared with other PHD domains. And the PHF6 was first identified as the gene mutated associated with Börjeson-Forssman-Lehmann syndrome, an X-linked mental retardation disorder. The mutant PHF6 is also associated with T cell acute lymphoblastic leukemia and acute myeloid leukemia. But the molecular mechanism between these diseases and PHF6 are still unclear. In addition, the first conserved ePHD (ePHD1) of PHF6 is involved in its nucleolus localization, directly interacts with upstream binding factor (UBF) and suppresses rRNA transcription. Here we show the backbone resonance and side chain assignments of the PHF6-ePHD1 domain from human by heteronuclear multidimensional NMR spectroscopy and its secondary structure as predicted by the TALOS+. These assignments of PHF6-ePHD1 domain throw a light on the further structure determination, dynamics and interaction with UBF.

Casein Kinase 1 α Phosphorylates the Wnt Regulator Jade-1 and Modulates Its Activity

Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner.

Functional analysis of a novel ENU-induced PHD finger 11 (Phf11) mouse mutant.

Previously, human genetic studies have shown association between polymorphisms within the gene encoding plant homeodomain zinc finger protein 11 (PHF11) and asthma-related phenotypes. Initial functional studies have suggested that PHF11 may be involved in the immune response through regulation of T cell activities. In order to study further the gene’s functions, we have investigated the mouse Phf11 locus. We have established and characterised a mouse line harbouring a point mutation in the PHD domain of Phf11. Full-length mouse cDNA for Phf11 was obtained by applying rapid amplification of cDNA ends (RACE). All five exons encoding the PHD domain of Phf11 were directly sequenced in 3840 mouse DNA samples from the UK MRC Harwell ENU (N-ethyl-N-nitrosourea)-mutagenised DNA archive. Mice harbouring a valine to alanine substitution, predicted to have a significant functional impact on the PHD zinc finger domain, were re-derived. These Phf11 mutant mice were outcrossed to C3H mice and then backcrossed for ten generations in order to establish a congenic line harbouring the single point mutation in Phf11. Macroscopic examination, haematology and histological examination of lung structure revealed no significant differences between mutant and wild-type mice. After administration of lipopolysaccharide, the level of expression of Il2, NF-kB and Setdb2 were significantly increased in Phf11 mutant homozygous lungs compared to control littermates. Our results provide evidence that Phf11 can operate as a Th1 cell regulator in immune responses. Moreover, our data indicate that these mice may provide a useful model for future studies on Phf11.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-014-9535-x) contains supplementary material, which is available to authorised users.

PHF11 is not a major candidate gene for asthma or eczema in Chinese children

Positional cloning and candidate gene studies in different Caucasian populations identified the gene encoding plant homeodomain zinc finger protein 11 (PHF11) to be associated with asthma and eczema. Microarray analysis also confirmed increased PHF11 expression in type 1 T-helper lymphocytes. However, such disease associations are unclear in Asian subjects. This case-control genetic association study investigated the relationship between asthma and eczema phenotypes and tagging single-nucleotide polymorphisms (SNPs) of PHF11 in Hong Kong Chinese children. Three hundred and nineteen asthmatic children and 236 children with eczema were recruited from hospital clinics and 445 children without any history of allergic disease were recruited as controls from local schools and hospitals. Atopy was defined by the presence of allergen-specific IgE in plasma or positive skin prick tests with wheal >or=3 mm larger than negative control. Lung function of asthmatics was evaluated by pre-bronchodilator spirometry. Ten PHF11 SNPs were genotyped by multiplex SNaPshot assay. Genotyping call rates were 100% for all SNPs, which also followed Hardy-Weinberg equilibrium. These SNPs were tightly linked in one haplotype block (D' >or= 0.95 for nearly all SNP pairs). Physician-diagnosed asthma was weakly associated with PHF11 +20860 and +22818 (P = 0.032 for both). Atopy was also associated with PHF11 +22398 (P = 0.029). However, none of the PHF11 SNPs was associated with eczema diagnosis and plasma total IgE and spirometric parameters in our patients. Our findings do not support PHF11 to be a major candidate gene for asthma, eczema and aeroallergen sensitization in Chinese children.

Specific Domain Structures Control Abscisic Acid-, Salicylic Acid-, and Stress-Mediated SIZ1 Phenotypes

SIZ1 (for yeast SAP and MIZ1) encodes the sole ortholog of mammalian PIAS (for protein inhibitor of activated STAT) and yeast SIZ SUMO (for small ubiquitin-related modifier) E3 ligases in Arabidopsis (Arabidopsis thaliana). Four conserved motifs in SIZ1 include SAP (for scaffold attachment factor A/B/acinus/PIAS domain), PINIT (for proline-isoleucine-asparagine-isoleucine-threonine), SP-RING (for SIZ/PIAS-RING), and SXS (for serine-X-serine, where X is any amino acid) motifs. SIZ1 contains, in addition, a PHD (for plant homeodomain) typical of plant PIAS proteins. We determined phenotypes of siz1-2 knockout mutants transformed with SIZ1 alleles carrying point mutations in the predicted domains. Domain SP-RING is required for SUMO conjugation activity and nuclear localization of SIZ1. Salicylic acid (SA) accumulation and SA-dependent phenotypes of siz1-2, such as diminished plant size, heightened innate immunity, and abscisic acid inhibition of cotyledon greening, as well as SA-independent basal thermotolerance were not complemented by the altered SP-RING allele of SIZ1. The SXS domain also controlled SA accumulation and was involved in greening and expansion of cotyledons of seedlings germinated in the presence of abscisic acid. Mutations of the PHD zinc finger domain and the PINIT motif affected in vivo SUMOylation. Expression of the PHD and/or PINIT domain mutant alleles of SIZ1 in siz1-2 promoted hypocotyl elongation in response to sugar and light. The various domains of SIZ1 make unique contributions to the plant's ability to cope with its environment.

Polymorphisms of PHF11 and DPP10 Are Associated with Asthma and Related Traits in a Chinese Population

Background: Initial studies by positional cloning have identified the genes encoding the plant homeodomain zinc finger protein 11 (PHF11) and dipeptidyl-peptidase 10 (DPP10) as asthma susceptibility genes. The variants in the two genes have been associated with asthma in several populations of European or Latin American origin. Objective: The aim of this study was to assess the common PHF11 and DPP10 polymorphisms for associations to asthma and asthma-related traits in a Han Chinese population. Methods: We genotyped six polymorphic markers in PHF11 and five polymorphic markers in DPP10 in a Han Chinese case-control cohort consisting of 408 asthma patients and 288 unrelated disease-free controls recruited from the Northern region of China. We analyzed the association between these markers and asthma as well as a number of intermediate, asthma-related traits. Linkage disequilibrium and haplotype patterns were also evaluated. Results: Significant associations were identified between two makers in PHF11 (rs1046295 and rs16659) and asthma susceptibility (odds ratio, OR = 1.32, 95% con fidence interval, CI = 1.06–1.65, p = 0.0096, for rs1046295, and OR = 1.41, 95% CI = 1.12–1.75, p = 0.0026, for rs16659). A strong association was observed between an SNP in DPP10 (rs10208402) and log_e-transformed total IgE (p = 0.0003) and the percentage of peripheral blood eosinophils (p = 0.0023). A weak association between rs1430090 in DPP10 and forced expiratory volume in 1 s was also observed (p = 0.048). Haplotype analysis revealed two protective haplotypes in PHF11 against asthma. Conclusion: The results provide supporting evidence for genetic variants in PHF11 and DPP10 genes underlying asthma susceptibility and asthma-related quantitative traits in a Han Chinese population.

The PHF11 gene is not associated with asthma or asthma phenotypes in two independent populations

Background:Numerous areas of the human genome have previously been associated with asthma and asthma-related phenotypes, but few positive findings have been successfully replicated in independent populations. Initial studies have reported...

Börjeson-Forssman-Lehmann Syndrome Due to a Novel Plant Homeodomain Zinc Finger Mutation in the PHF6 Gene

The Börjeson-Forssman-Lehmann syndrome is an X-linked mental retardation disorder caused by mutations in the PHF6 gene. The PHF6 gene contains 2 plant homeodomain zinc fingers, suggesting a role for the protein in chromatin remodeling. In this study, the authors report on a Finnish family with a classical Börjeson-Forssman-Lehmann syndrome phenotype caused by a G to T nucleotide substitution at position 266 within exon 4 within the PHF6 gene (c.266G>T). The resulting glycine to valine (p.G89V) change corresponds to a highly conserved residue within the first plant homeodomain zinc finger domain. This is a novel change that adds to the number of plant homeodomain zinc finger mutations identified, such that 23% of all Börjeson-Forssman-Lehmann syndrome mutations lie within this motif. Moreover, it highlights the functional importance of plant homeodomain zinc finger motifs to human disease and more specifically to PHF6 function.

The Polybasic Region That Follows the Plant Homeodomain Zinc Finger 1 of Pf1 Is Necessary and Sufficient for Specific Phosphoinositide Binding

The plant homeodomain (PHD) zinc finger is one of 14 known zinc-binding domains. PHD domains have been found in more than 400 eukaryotic proteins and are characterized by a Cys(4)-His-Cys(3) zinc-binding motif that spans 50-80 residues. The precise function of PHD domains is currently unknown; however, the PHD domains of the ING1 and ING2 tumor suppressors have been shown recently to bind phosphoinositides (PIs). We have recently identified a novel PHD-containing protein, Pf1, as a binding partner for the abundant and ubiquitous transcriptional corepressor mSin3A. Pf1 contains two PHD zinc fingers, PHD1 and PHD2, and functions to bridge mSin3A to the TLE1 corepressor. Here, we show that PHD1, but not PHD2, binds several monophosporylated PIs but most strongly to PI(3)P. Surprisingly, a polybasic region that follows the PHD1 is necessary for PI(3)P binding. Furthermore, this polybasic region binds specifically to PI(3)P when fused to maltose-binding protein, PHD2, or as an isolated peptide, demonstrating that it is sufficient for specific PI binding. By exchanging the polybasic regions between different PHD fingers we show that this region is a strong determinant of PI binding specificity. These findings establish the Pf1 polybasic region as a phosphoinositide-binding module and suggest that the PHD domains function down-stream of phosphoinositide signaling triggered by the interaction between polybasic regions and phosphoinositides.

Role for the mortality factors MORF4, MRGX, and MRG15 in transcriptional repression via associations with Pf1, mSin3A, and Transducin-Like Enhancer of Split.

mSin3A and Transducin-Like Enhancer of Split (TLE) are two histone deacetylase (HDAC)-containing corepressors that function to repress transcription at targeted genes. Pf1 is a plant homeodomain zinc finger protein that interacts with both mSin3A and TLE, suggesting that it coordinates their function. Here we show that mSin3A and TLE interact with members of the mortality factor (MORF) family of putative transcriptional regulators. This family comprises MORF on chromosome 4 (MORF4) and MORF-related genes on chromosomes X and 15 (MRGX and MRG15, respectively) and is proposed to contribute to cellular senescence. Consistent with a role in transcription, we demonstrate that Gal4 fusions to each MORF family member repress transcription from a Gal4-dependent luciferase reporter. By using both mapping experiments and a dominant negative form of TLE, we show that repression by MORFs requires associations with mSin3A and TLE. Therefore, common functions of the MORFs are likely elicited through the action of a MORF/mSin3A/TLE complex. While the MORFs may have common functions, MRG15, but not MRGX or MORF4, interacted with Pf1. Therefore, MRG15 may have functions that are distinct from those of MRGX and MORF4. Consistent with this hypothesis, Pf1 reduced transcriptional repression by Gal4-MRG15 but it had no effect on repression by MRGX and MORF4. Pf1 has independent binding sites for MRG15 and mSin3A. In addition, Pf1 and MRG15 bind different domains on mSin3A. Together, these data suggest that the unique functions of MRG15 are elicited through the action of an MRG15/Pf1/mSin3A complex.

Genetic characterization of Drosophila Mi-2 ATPase

Mammalian Mi-2, an auto-antigen for dermatomyositis, is known to be an adenosine triphosphate (ATP)-dependent nucleosome remodelling factor. The Drosophila homologue of Mi-2 (dMi-2) gene is located at 76D5-6 on the left arm of the third chromosome and is transcribed into two alternate transcripts (dMi-2a and dMi-2b). Both transcripts are present at high levels in the ovary and during the first 8 h of embryogenesis when detected by Northern blot analysis. The localization of protein was nuclear, which is consistent with its proposed function as a component of the chromatin remodelling complex. Several lines of recessive mutants including mutations in dMi-2 were isolated and classified into four different complementation groups. Four alleles of dMi-2 mutants were further characterized in molecular nature; dMi-2BL1 was found to have a mutation in the ATP-binding motif of the ATPase domain, dMi-2BL7 in the core histidine of the first plant homeodomain zinc finger and dMi-2BL12 in a conserved serine in the chromodomain. On the other hand, dMi-2BL3 did not have any change in the coding region. The expression pattern of dMi-2 and the embryonic lethal phenotypes of mutants indicate that dMi-2 is essential for embryonic development in Drosophilamelanagaster.

Functional Organization of MIR2, a Novel Viral Regulator of Selective Endocytosis

Kaposi's sarcoma-associated herpesvirus encodes two related proteins, MIR1 and MIR2, that lead to reduction of the cell surface levels of major histocompatibility complex class I and other polypeptides involved in immune recognition. MIR1 and MIR2 do not affect the assembly or transport of their target proteins through the secretory pathway; rather, they act to enhance the selective endocytosis of target chains from the cell surface. Sequence inspection reveals that the modulator of immune recognition (MIR) proteins contain an NH(2)-terminal zinc finger of the plant homeodomain (PHD) subfamily, two transmembrane (TM) domains, and a C-terminal conserved region (CR). Here we examine the transmembrane topology and functional organization of MIR2. Both the PHD domain and the CR are disposed cytosolically and are essential for MIR-mediated endocytosis. MIR proteins form homo-oligomers; this activity is independent of the PHD and CR elements and maps instead to the TM regions. Analysis of chimeras between MIR1 and MIR2 reveals that the TM regions also mediate target selectivity. Mutations that ablate the PHD or CR regions generate dominant negative phenotypes for major histocompatibility complex class I endocytosis. These findings suggest a domain organization for the MIR proteins, with the TM regions involved in target selection and the cytosolic PHD and CR domains involved in the possible recruitment of cellular machinery that directly or indirectly regulates internalization of target molecules.

The Autoimmune Regulator (AIRE) Is a DNA-binding Protein

The autoimmune regulator (AIRE) protein is a putative transcription regulator with two plant homeodomain-type zinc fingers, a putative DNA-binding domain (SAND), and four nuclear receptor binding LXXLL motifs. We have shown here that in vitro, recombinant AIRE can form homodimers and homotetramers that were also detected in thymic protein extracts. Recombinant AIRE also oligomerizes spontaneously upon phosphorylation by cAMP dependent protein kinase A or protein kinase C. Similarly, thymic AIRE protein is phosphorylated at the tyrosine and serine/threonine residues. AIRE dimers and tetramers, but not the monomers, can bind to G-doublets with the ATTGGTTA motif and the TTATTA-box. Competition assays revealed that sequences with one TTATTA motif and two tandem repeats of ATTGGTTA had the highest binding affinity. These findings demonstrate that AIRE is an important DNA binding molecule involved in immune regulation.

Pf1, a novel PHD zinc finger protein that links the TLE corepressor to the mSin3A-histone deacetylase complex.

The mSin3A-histone deacetylase corepressor is a multiprotein complex that is recruited by DNA binding transcriptional repressors. Sin3 has four paired amphipathic alpha helices (PAH1 to -4) that are protein-protein interaction motifs and is the scaffold upon which the complex assembles. We identified a novel mSin3A-interacting protein that has two plant homeodomain (PHD) zinc fingers we term Pf1, for PHD factor one. Pf1 associates with mSin3A in vivo and recruits the mSin3A complex to repress transcription when fused to the DNA binding domain of Gal4. Pf1 interacts with Sin3 through two independent Sin3 interaction domains (SIDs), Pf1SID1 and Pf1SID2. Pf1SID1 binds PAH2, while Pf1SID2 binds PAH1. Pf1SID1 has sequence and structural similarity to the well-characterized 13-amino-acid SID of the Mad bHLHZip repressor. Pf1SID2 does not have sequence similarity with either Mad SID or Pf1SID1 and therefore represents a novel Sin3 binding domain. Mutations in a minimal fragment of Pf1 that encompasses Pf1SID1 inhibited mSin3A binding yet only slightly impaired repression when targeted to DNA, implying that Pf1 might interact with other corepressors. We show that Pf1 interacts with a mammalian homolog of the Drosophila Groucho corepressor, transducin-like enhancer (TLE). Pf1 binds TLE in an mSin3A-independent manner and recruits functional TLE complexes to repress transcription. These findings suggest that Pf1 may serve to bridge two global transcription networks, mSin3A and TLE.

Autoantibodies against Aromatic l-Amino Acid Decarboxylase Identifies a Subgroup of Patients with Addison’s Disease1

Autoantibodies against aromatic L-amino acid decarboxylase (AADC) are present in about 50 percent of sera from patients with autoimmune polyendocrine syndrome type I (APS I) but absent in sera from patients with different organ-specific autoimmune diseases, such as insulin-dependent diabetes mellitus, Hashimoto's thyroiditis, and Graves' disease. AADC is expressed in the pancreatic beta-cells, the liver, and the nervous system; and the presence of AADC antibodies has been shown to correlate to hepatitis and vitiligo in APS I patients. Among 101 investigated patients with autoimmune Addison's disease, 15 had high titers of AADC antibodies. According to the clinical characteristics of these patients, only 3 had APS I. The remaining 12 had either isolated Addison's disease or associated diabetes mellitus, hypothyroidism, vitiligo, alopecia, gonadal failure, and pernicious anemia. Autoantibodies against 21-hydroxylase were present in 9 of 12, whereas autoantibodies against side-chain cleavage enzyme and 17alpha-hydroxylase were present in 3 of 12. Two patients had only autoantibodies against AADC. DNA was available from 3 of these 12 patients. One of the patients, a woman with Addison's disease, autoimmune thyroiditis, and premature menopause was heterozygous for a point mutation (G1021A, Val301Met) in the first plant homeodomain zinc finger domain of the autoimmune regulator (AIRE) gene. The presence of AADC autoantibodies identifies patients with APS I and a subgroup of Addison patients who may have a milder atypical form of APS I or represent a distinct entity. Measurement of autoantibodies against AADC should be included in the evaluation of Addison's disease.