Genes Encoding Zinc Finger Proteins Research Articles

Modulation of the wheat transcriptome by TaZFP13D under well-watered and drought conditions

Maintaining global food security in the context of climate changes will be an important challenge in the next century. Improving abiotic stress tolerance of major crops such as wheat can contribute to this goal. This can be achieved by the identification of the genes involved and their use to develop tools for breeding programs aiming to generate better adapted cultivars. Recently, we identified the wheat TaZFP13D gene encoding Zinc Finger Protein 13D as a new gene improving water-stress tolerance. The current work analyzes the TaZFP13D-dependent transcriptome modifications that occur in well-watered and dehydration conditions to better understand its function during normal growth and during drought. Plants that overexpress TaZFP13D have a higher biomass under well-watered conditions, indicating a positive effect of the protein on growth. Survival rate and stress recovery after a severe drought stress are improved compared to wild-type plants. The latter is likely due the higher activity of key antioxidant enzymes and concomitant reduction of drought-induced oxidative damage. Conversely, down-regulation of TaZFP13D decreases drought tolerance and protection against drought-induced oxidative damage. RNA-Seq transcriptome analysis identified many genes regulated by TaZFP13D that are known to improve drought tolerance. The analysis also revealed several genes involved in the photosynthetic electron transfer chain known to improve photosynthetic efficiency and chloroplast protection against drought-induced ROS damage. This study highlights the important role of TaZFP13D in wheat drought tolerance, contributes to unravel the complex regulation governed by TaZFPs, and suggests that it could be a promising marker to select wheat cultivars with higher drought tolerance.

Cell Surface Proteins of B Cells and Plasmacells Are Differently Expressed in Waldenström’s Macroglobulinemia (WM) Patients Vs. Subjects with Monoclonal Gammopathy of Uncertain Significance (IgMMGUS) Vs. Healthy Donors

We performed gene expression profiling (GEP) of 36 patients with Waldenström's Macroglobulinemia (WM), 13 subjects with Monoclonal Gammopathy of Uncertain Significance (IgMMGUS), and 7 healthy subjects (CTRLs) by Affymetrix GeneChip Human Genome U133 Plus 2.0. GEP was performed on 36 WM bone marrow (BM) CD19+ cells vs. 10 IgMMGUS BM CD19+cells vs. 7 CTRLs BM CD19+ cells. We analyzed 32 WM BM CD138+ cells vs. 10 IgMMGUS BM CD138+ cells vs. 7 CTRLs BM CD138+ cells by microarray. Data was preprocessed and normalized using RMA and ComBat and analyzed using Statistical Analysis for Microarrays (SAM) on 3 groups and a false discovery rate threshold of 5%, followed by a pair-wise SAM test. We found 2038 and 29 unique genes in the comparison between WM, IgMMGUS and CRTLs in CD19+ and CD138+ cells, respectively. There were 16 differently expressed (DE) genes in common between the CD19+ and the CD138+ cells. Genes were grouped accordingly to the following patterns of differential expression: 1) WM>MGUS>CTRL, 2) WM<MGUS<CTRL, 3) WM>MGUS but MGUS<CTRL, 4) WM<MGUS but MGUS>CTRL (Tab.1). Functional enrichment analysis and annotation clustering (DAVID) were run on each pattern. We demonstrated gene expression changes between WM, IgMMGUS and CTRLs in genes encoding hematopoietic cell lineage markers. We showed thatIL2RA, CD1D,andCD44were over expressed in WM vs. IgMMGS, and WM vs. CTRLs.CD25expression characterizes Waldenström's clone (García-Sanz et. al. Best Pract Res Clin Haematol 2016). The over expression ofCD1Don WM B cells compared to IgMMGUS and CTRLs, suggests its possible role as a marker in WM as previously shown in B-cell chronic lymphoproliferative disorders (Kotsianidis et. al. Am J Clin Pathol 2011). We found thatCD44,encoding a glycoprotein over expressed in cancer stem cells, was up regulated in WM B cells compared to the same cell counterpart of IgMMGUS and CTRLs (Chen et. al. J Hematol Oncol 2018). Functional clustering identified several genes encoding cell adhesion molecules (CAM) which are expressed on the surface of B lymphocytes and are involved in the binding with other cells, immune response (T cells), and inflammation. Class I histocompatibility genesHLA-A, HLA-C, HLA-B, HLA-E,andHLA-Gwere over expressed in WM vs. IgMMGUS, and WM vs. CTRLs. Previous studies demonstrated the higher levels of HLA-Gs in WM and IgMMGUS compared to CTRL. Moreover, HLA-G could represent a marker of clonality since high levels of HLA-Gs were observed in both IgG and IgA MGUS and MM patients (Moreau et. al. EJH 2008). CNTNAP2andADAM23were down regulated in WM vs. CTRLs.CNTNAP2,encoding CASPR2 protein, is expressed in CNS and PNS and is involved in neurodevolpmental disorders and the autoimmune disease Sjogren's syndrome (SS) (Xiao Clin Rheumatol 2017). In addition, Caspr2 and ADAM23 take part in the peripheral neuropathy by controlling the activity of potassium channels in neurological autoimmune diseases (Saint-Martin et al. Eur J Neurosci 2018). We can speculate the role of both genes in WM patients with Sjogren's syndrome. VWF(von Willebrand factor),TFPI, andSERPINE1belonging to the complement and coagulation cascade were under expressed in WM vs. IgMMGUS. Notably, some authors have demonstrated that a low level of VWF was associated with high serum IgM levels and withCXCR4mutations in WM patients, and that response to treatment improves VWF in WM patients (Castillo et. al. BJH 2019). Genes encoding zinc finger proteins (ZNF) (n=120) were deregulated in the comparison between WM vs. IgMMGUS, and vs. CTRLS CD19+ cells in all patterns. We suggest the possible role of ZNF in the transcription regulation and cancer progression in the comparison between the 3 groups of subjects. Of note,ZNF804AandZNF215were upregulated in WM vs. IgMGUS and WM vs. CTRLs with high FC (Guerrera et. al. Haemat 2018). TJP1was under expressed in WM CD19+ and WM CD138+ cells. We noted very high FC in CD138+ cells: WM vs. IgMMGUS (FC=-2,65), and WM vs. CTRLs (FC=-6,18). HighTJP1expression in myeloma plasma cells was associated to a better and longer response to protease Inhibitors (PI) suggesting this gene as a biomarker to identify patients which can benefit from PI-based therapy (Zhang et. al. Cancer Cell 2016). Our findings demonstrate that B cells show different gene expression levels in membrane adhesion and cell lineage pathways in WM compared to IgMMGUS and CTRLs, suggesting the possible role of these genes in the progression of IgMMGUS to WM. Disclosures Tedeschi: AstraZeneca:Consultancy, Membership on an entity's Board of Directors or advisory committees;Janssen spa:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;AbbVie:Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;BeiGene:Consultancy, Membership on an entity's Board of Directors or advisory committees;Department of Hematology Niguarda Hospital Milano:Current Employment;Sunesis:Consultancy.Montillo:F. Hoffmann-La Roche:Honoraria, Research Funding;Astra Zeneca:Honoraria;AbbVie:Honoraria, Speakers Bureau;Janssen:Honoraria, Speakers Bureau;Verastem:Honoraria;Gilead:Honoraria, Speakers Bureau.

ZNF804A Gene Variants Have a Cross-diagnostic Influence on Psychosis and Treatment Improvement in Mood Disorders.

ObjectiveGenetic variations in the gene encoding zinc finger protein 804A gene (ZNF804A) have been associated with major depression and bipolar disorder. In this work we focused on the potential influence of ZNF804A variations on the risk of developing specific sub-phenotypes as well as the individual response to available treatments.MethodsWe used two samples of different ethnic origin: a Korean sample, composed by 242 patients diagnosed with major depression and 132 patients diagnosed with bipolar disorder and 326 healthy controls; an Italian sample composed 151 major depression subjects, 189 bipolar disorder subjects and 38 outpatients diagnosed for a primary anxiety disorder.ResultsOur analyses reported an association of rs1344706 with psychotic phenotype in the cross-diagnostic pooled sample (geno p = 4.15 × 10−4, allelic p = 1.06 × 10−4). In the cross-diagnosis Italian sample but not in the Korean one, rs7597593 was involved with depressive symptoms improvement after treatment (geno p = 0.025, allelic p = 0.007).ConclusionThe present study evidenced the role of ZNF804A alterations in symptoms improvement after treatment. Both manic and depressive symptoms seem to be modulated by ZNF804A, though the latter was observed in the bipolar pooled sample only. The role of this factor is likely related to synaptic development and maintenance; however, further analyses will be needed to better understand the molecular mechanics involved with ZNF804A.

Whole-exome sequencing in familial keratoconus: the challenges of a genetically complex disorder.

The underlying genetic causes of keratoconus are essentially unknown. Here, we conducted whole-exome sequencing in 2 Brazilian families with keratoconus. Whole-exome sequencing was performed on 6 keratoconus-affected individuals of 2 unrelated pedigrees from Southern Brazil. Pathogenic variants were identified in a modified Trio analysis (1 parent and 2 children) using candidate gene filtering. All the affected subjects underwent detailed corneal tomographic evaluation. Clinically relevant variants that were present in affected individuals at minor allele frequencies <1% were examined in the 1000 Genomes Project single nucleotide polymorphism ABraOM and transcription gene (RefSeq and Ensembl) databases. In family 1, a sequence variant in chromosome 1 (q21.3) was observed within the filaggrin gene. All the tested family members shared a heterozygous missense pathogenic variant in the c.4678C>T position. In family 2, exome analysis demonstrated a sequence variant in chromosome 16 (q24.2) within the gene encoding zinc finger protein 469 (ZNF469). Members of family 2 shared a heterozygous missense variant in the c.1489G>A position. In addition, the exomes of the 2 families were examined for shared genetic variants among all affected individuals. Filtering criteria did not identify any rare sequence variants in a single gene segregated in both families. Our findings show that a complete genotype-phenotype correlation could not be identified, suggesting that keratoconus is a genetically heterogeneous disease. In addition, we believe that whole-exome sequencing-based segregation analysis is probably not the best strategy for identifying variants in families with isolated keratoconus.

Transcriptome and Resistance-Related Genes Analysis of Botrytis cinerea B05.10 Strain to Different Selective Pressures of Cyprodinil and Fenhexamid.

The pathogen Botrytis cinerea is a very dangerous pathogen that infects many economically important crops such as grape, strawberry, tomato, and eggplant. Cyprodinil, a pyrimidine amine fungicide, and fenhexamid, an amide fungicide, are new reagents for controlling gray mold with special efficacy. It is necessary to understand the change trends in the toxicological and physiological characteristics of B. cinerea with successive selective pressures of cyprodinil and fenhexamid to elongate the serving life of these fungicides for effective disease control. The toxicities of cyprodinil and fenhexamid at successive concentrations of EC25, EC50 and EC75 on B. cinerea strain BO5.10 were assayed along with mycelial growth-inhibition capacity. The results showed that the EC50 value of the cyprodinil-treated F27 strain increased approximately 18-fold, whereas of which in the fenhexamid-treated F27 strain increased only 3-fold compared with that of the F0 strain. The conductivities and glycerinum contents of the strains resistant to cyprodinil and fenhexamid were obviously enhanced; in contrast, the oxalic acid contents were decreased compared with those in the F0 strain. The transcriptomes of the F27 control (T01), cyprodinil-treated (T02) and fenhexamid- treated (T03) strains were analyzed, and the expression levels of functional genes in the T02 and T03 strains were significantly increased compared with those in the T01 strain; these results were further validated using qRT-PCR. The results indicated that the relative expression of two genes encoding mixed-functional oxidases (MFOs) BC1G_16062 and BC1G_16084, two genes encoding transmembrane proteins BC1G_12366 and BC1G_13768, two genes encoding Zinc finger proteins BC1G_13764 and BC1G_10483,one gene encoding citrate synthase enzyme BC1G_09151, one gene encoding gluconolactonase BC1G_15612 in the T02 and T03 strains and one gene encoding lysophospholipids enzyme BC1G_04893 in the T3 strain increased substantially compared with that in the T1 strain (P < 0.01). Functional prediction analysis of upregulated gene expression and structural verification was also performed, and the results showed that BC1G_10483 was a ZnF_C2HC transcriptional regulator interacting with the Sp1 element of these genes to respond to the pressures from cyprodinil and fenhexamid. Our results could contribute to a better understanding of the resistance mechanism of B. cinerea against cyprodinil and fenhexamid.

Genetic influence on xanthine oxidase activity and arginine metabolites in Hispanic children

Xanthine oxidase (XO) catalyzes the oxidation of xanthine and hypoxanthine in the uric acid metabolic pathway generating reactive oxygen species in the process. Inhibition of XO activity is known to reduce oxidative damage and improve endothelial function. The aim of this study was to identify the genetic basis of estimated XO activity and its relationship with endothelial function biomarkers in 752 Hispanic children of the Viva La Familia Study. Xanthine, hypoxanthine and other biomarkers were measured as part of metabolomics profiling. Xanthine oxidase activity was estimated as a product/precursor ratio. Quantitative genetic analysis was conducted using a variance components decomposition‐based method accounting for kinships. Significant heritabilities were obtained for XO activity (h2 = 0.60 ±0.08; p = 1.6 × 10−14), homocysteine (h2 = 0.82± 0.07; p = 5.2 × 10−32) and arginine metabolites; arginine (h2 = 0.47± 0.09; p = 4.6 × 10−9) and dimethylarginine (h2 = 0.24 ± 0.08; p = 0.001). Significant genetic and phenotypic correlations were found between XO activity and homocysteine (rhog = −0.21 ± 0.10; p &lt; 0.05; rhop = −0.15 ± 0.05; p &lt; 0.0005), arginine (rhog = 0.39 ± 0.13; p &lt; 0.005; rhop = 0.15 ± 0.05; p &lt; 0.0005) and dimethylarginine (rhog = −0.60 ± 0.17; p &lt; 0.0005; rhop = −0.13 ± 0.05; p &lt; 0.001). Genome‐wide association analysis showed significant association of XO with rs7809463 in archaelysin family metallopeptidase1 (AMZ1) and rs58632700 in a gene encoding zinc finger protein 446 (ZNF446) (p &lt; 10−8). Follow up bivariate analysis found significant evidence of pleiotropic effects of echinoderm microtubule associated protein like 6 (EML6) on XO activity and methylarginine (p &lt; 10−8), and suggestive evidence of AMZ1 and ZNF446 on XO activity and arginine (p &lt; 10−7). Interestingly, L‐Arginine is known to have a protective effect against reactive oxygen species and its supplementation is believed to reduce oxidative damage. The results show that shared genetic factors influence XO activity and arginine metabolites with opposing effects in Hispanic children with important implications for diseases such as obesity, diabetes and cardiovascular disease.Support or Funding InformationNational Institutes of Health (NIH) [DK080457], and the USDA/ARS [Cooperative Agreement6250‐51000‐053]

ZNF804A Genetic Variation Confers Risk to Bipolar Disorder

Genetic variation in the gene encoding zinc finger protein 804A gene (ZNF804A) has been identified to be associated with bipolar disorder in genome-wide association studies, while little is known regarding their association in Asian populations. In this study, we aimed to examine whether ZNF804A may confer susceptibility to BD in Han Chinese. We measured the mRNA expression level of ZNF804A in patients with BD and controls. Two functional variants (rs1344706 and rs359895) in ZNF804A were genotyped among 1508 individuals with or without BD. We then performed a meta-analysis based on present literature. Our results showed that ZNF804A mRNA level in peripheral blood mononuclear cells was significantly higher in BD patients than that in controls (P = 0.01). The rs1344706 showed significant allelic association with BD (P = 0.034). The frequency of T allele of rs1344706 was higher in patients than that in controls (odds ratio = 1.19, 95% confidence interval 1.03-1.37). After pooling all data, meta-analysis indicated a significant association of rs1344706 with BD (P = 0.0003). Furthermore, we performed an eQTL analysis and observed significant associations between rs1344706 and ZNF804A expression level in hippocampus (P = 0.032) and occipital cortex (P = 0.036). After stratified by diagnosis, the association of rs1344706 with BD was only survived in BD-I (genotypic P = 0.04, allelic P = 0.014), but not in BD-II. Our findings provided supportive evidence for the involvement of ZNF804A rs1344706 in BD, especially in BD-I.

Balanced translocation t(3;18)(p13;q22.3) and points mutation in the ZNF407 gene detected in patients with both moderate non-syndromic intellectual disability and autism

Intellectual disability (ID) is a common disease. While the etiology remains incompletely understood, genetic defects are a major contributor, which include mutations in genes encoding zinc finger proteins. These proteins modulate gene expression via binding to DNA. Consistent with this knowledge, we report here the identification of mutations in the ZNF407 gene in ID/autistic patients. In our study of an ID patient with autism, a reciprocal translocation 46,XY,t(3;18)(p13;q22.3) was detected. By using FISH and long-range PCR approaches, we have precisely mapped the breakpoints associated with this translocation in a gene-free region in chromosome 3 and in the third intron of the ZNF407 gene in chromosome18. The latter reduces ZNF407 expression. Consistent with this observation, in our subsequent investigation of 105 ID/autism patients with similar clinical presentations, two missense mutations Y460C and P1195A were identified. These mutations cause non-conservative amino acid substitutions in the linker regions between individual finger structures. In line with the linker regions being critical for the integrity of zinc finger motifs, both mutations may result in loss of ZNF407 function. Taken together, we demonstrate that mutations in the ZNF407 gene contribute to the pathogenesis of a group of ID patients with autism.

Systematic analysis of gene expression level with tissue-specificity, function and protein subcellular localization in human transcriptome

Recent studies have shown that, in mammals, the highly expressed genes have shorter gene length and their protein products have relatively lower evolutionary rates. However, the global relationship between genes' expression level and their features such as tissue-specificity, function and protein subcellular localization has not been investigated extensively, especially in mammalian. In order to solve it, we analysed 8,570 genes across 46 human tissues. Our results suggest that widely expressed genes have higher mean expression levels than tissue-specific ones and genes encoding zinc-finger proteins have low expression levels similar to that of DNA-binding proteins. In the analysis of protein subcellular localization, it is shown that nuclear and Golgi apparatus proteins have lower mean expression levels than those of mitochondria, endoplasmic reticulum and membrane proteins, while genes encoding cytoplasm and extracellular components display the highest expression levels. When comparing the gene expression levels and the number of expressed genes in different tissues, we found that some tissues have less active genes while single gene encodes relatively more transcripts. Taken together, gene expression levels are clearly correlated with their tissue-specificity, function and protein subcellular localization, and are highly conserved during evolution.

ZFP191 is required by oligodendrocytes for CNS myelination

The controlling factors that prompt mature oligodendrocytes to myelinate axons are largely undetermined. In this study, we used a forward genetics approach to identify a mutant mouse strain characterized by the absence of CNS myelin despite the presence of abundant numbers of late-stage, process-extending oligodendrocytes. Through linkage mapping and complementation testing, we identified the mutation as a single nucleotide insertion in the gene encoding zinc finger protein 191 (Zfp191), which is a widely expressed, nuclear-localized protein that belongs to a family whose members contain both DNA-binding zinc finger domains and protein-protein-interacting SCAN domains. Zfp191 mutants express an array of myelin-related genes at significantly reduced levels, and our in vitro and in vivo data indicate that mutant ZFP191 acts in a cell-autonomous fashion to disrupt oligodendrocyte function. Therefore, this study demonstrates that ZFP191 is required for the myelinating function of differentiated oligodendrocytes.

Association of HOXA10, ZFPM2, and MMP2 genes with scrotal hernias evaluated via biological candidate gene analyses in pigs

To evaluate the associations between 14 biological candidate genes and scrotal hernias in pigs. 1,534 Pietrain-based pigs, including 692 individuals from 298 pig families and 842 male pigs without family information. Pigs were classified as affected or unaffected for scrotal hernias. Single nucleotide polymorphisms of candidate genes were analyzed via PCR assays and genotyped. Statistical analyses were performed on the family-trio and the case-control data. 2 genes involved in collagen metabolism (homeobox A10 [HOXA10] and matrix metalloproteinases 2 [MMP2]) and 1 gene encoding zinc finger protein multitype 2 (ZFPM2, important in the development of diaphragmatic hernia) were significantly associated with hernias. Pigs with these genotypes had high odds of developing scrotal hernias in the case and control groups (2 ZFPM2 variants: odds ratio, 4.3 [95% confidence interval, 2.78 to 6.64] and 4.45[95%confidenceinterval,2.88to6.88]). Anothergene, collagentypeII A 1(COL2A1),was potentially involved in hernia development. HOXA10, ZFPM2, MMP2, and COL2A1 could have important roles in pig hernia development and potentially be useful for marker-assisted selection in the pig industry. Pigs are used for the study of many human diseases because of their physiologic similarities. Genes associated with scrotal hernias in this study may be directly used in understanding the molecular mechanisms underlying this defect in humans.

Molecular cloning and characterization of a novel gene encoding zinc finger protein from Medicago sativa L.

A suppression subtraction hybridization (SSH) cDNA library had been constructed to identify differentially expressed genes. Based on the sequence of an expressed sequence tag (EST) homologous to Pisum sativum zinc finger protein mRNA (Accession number: AF160911), the full-length cDNA of 1,676 nucleotides was cloned from alfalfa by rapid amplification of cDNA ends (RACE). It was designated as MsZFN, encoding a protein of 418 amino acids. The amino acid sequence compared by blast revealed high homology with zinc finger protein of other plants. Sequence comparison showed that there were five conserved typical zinc finger motifs, and one sugar transfer protein signature. The calculated molecular weight of the MsZFN protein was 45.8 k Da, and theoretical isoelectric point was 8.13. The MsZFN localized in nucleus. Under normal growth conditions, differential expression of MsZFN exhibited that the expression was the highest in leaf and the lowest in root. MsZFN was quickly and transiently induced by NaCl treatment and reached its maximum at 30 min.

Zic3z defines the dorsal and vegetal neuroectoderm in the zebrafish embryonic development

The Zic family is a group of genes encoding zinc finger proteins that are highly expressed in the mammalian cerebellum. Zic genes are the vertebrate homologue of Drosophila pair‐rule gene, odd‐paired (opa), which plays important roles in the parasegmental subdivision as well as in the visceral mesoderm development of Drosophila embryos. Recent studies on human, mouse, frog, fish and ascidian Zic homologues support that Zic genes are involved in a variety of developmental processes, including neurogenesis, myogenesis, skeletal patterning, and left‐right axis establishment. In an effort to explore possible functions of Zic proteins during vertebrate embryogenesis, we initially examined more detailed expression pattern of zebrafish homologue of zic3 (zic3z). zic3z transcripts are detected in the neuroectoderm, neural plate, dorsal neural tube, and brain regions including eye field during early embryonic development. Marker DNA studies found that zic3z transcription is modulated by BMP, Wnt, and Nodal signals particularly in the dorsal and vegetal neuroectoderm at gastrula. Interfering with zic3z translation with zic3z‐specific morpholino causes abnormal brain formation and expansion of the optic stalk cells. Retinal ganglion cells (RGCs) undergo abnormal neuronal differentiation. These findings suggest that zic3z defines the dorsal and vegetal neuroectoderm to specify brain formation and retinal neurogenesis during early embryonic development.

Mapping of MYC breakpoints in 8q24 rearrangements involving non-immunoglobulin partners in B-cell lymphomas

Chromosomal translocations joining the immunoglobulin (IG) and MYC genes have been extensively reported in Burkitt's and non-Burkitt's lymphomas but data concerning MYC rearrangements with non-IG partners are scarce. In this study, 8q24 breakpoints from 17 B-cell lymphomas involving non-IG loci were mapped by fluorescence in situ hybridization (FISH). In seven cases the breakpoint was inside a small region encompassing MYC: in one t(7;8)(p12;q24) and two t(3;8)(q27;q24), it was telomeric to MYC whereas in four cases, one t(2;8)(p15;q24) and three t(8;9)(q24;p13) it was located in a 85 kb region encompassing MYC. In these seven cases, partner regions identified by FISH contained genes known to be involved in lymphomagenesis, namely BCL6, BCL11A, PAX5 and IKAROS. Breakpoints were cloned in two t(8;9)(q24;p13), 2.5 and 7 kb downstream from MYC and several hundred kb 5' to PAX5 on chromosome 9, joining MYC to ZCCHC7 and to ZBTB5 exon 2, two genes encoding zinc-finger proteins. In these seven cases, MYC expression measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) was significantly higher when compared to that of patients without 8q24 rearrangement (P=0.006). These results suggest that these rearrangements are the consequence of a non-random process targeting MYC together with non-IG genes involved in lymphocyte differentiation and lymphoma progression.

Identification of Switching-Related Transcription Factors from a Comparison of Cord and Adult Human Blood Reticulocytes Transcriptomes.

Hemoglobin switching patterns during human ontogeny are highly correlated with transcription of the alpha and beta globin genes on chromosomes 16 and 11, but the molecular and genetic mechanisms responsible for the switching phenomenon are not yet defined. For a better understanding of post-natal globin gene switching and silencing, the expression profiles from purified reticulocyte mRNA (28 separate clinical samples; 14 cord, 14 adult) were studied using Affymetrix HG-U133 arrays (44,229 probe sets). To validate the transcriptome expression levels derived from the microarrays and to discriminate the differences between cord blood and adult blood reticulocytes, we performed quatitative real-time PCR. Among 21 tested control genes, the ratios of adult-to-cord blood (AB/CB) array hybridization intensities versus PCR copy numbers demonstrated a correlation coefficient of 0.978. Hence, the AB/CB array hybridization intensities provide a reliable description of erythroid transcriptome patterns associated with post-natal hemoglobin switching. Stringent screening criteria were then used to identify genes that may be involved in fetal-to-adult hemoglobin switching. All 44,229 probe sets were filtered to identify genes that demonstrated greater than a five fold difference in AB and CB mean signal intensities as well as consistent differences among the 14 samples from each group. In addition, the screening criteria required high-level signals in either AB or CB (>1000 FU), and t-test p-values between AB and CB <0.0001. 107 switching-related probe sets from 94 genes met these criteria, and over 90% of them were detected at higher levels in CB. When this inventory of probe sets was categorized using Gene Ontology, less than half were assigned a known molecular function. Among those, five transcription factors were identified including LIM domain only 2 (LMO2) and Kruppel like factor 3 (KLF3). Those two transcription factors play known roles in hematopoiesis, and LMO2 is required for embryonic erythropoiesis. Two other genes encode zinc finger proteins, but no description of their expression in hematopoietic tissues has been reported. Finally, a novel gene encoding chromatin remodeling and PHD-finger domains was discovered. Postnatal hemoglobin switching is associated with significant changes in the reticulocyte transcriptome. Among the transcripts demonstrating highly significant shifts in their levels, known and novel transcription factors were identified.

Mouse Zic5 deficiency results in neural tube defects and hypoplasia of cephalic neural crest derivatives

Zic family genes encode zinc finger proteins, which are homologues of the Drosophila pair-rule gene odd-paired . In the present study, we characterized the fifth member of the mouse Zic family gene, mouse Zic5 . Zic5 is located near Zic2 , which is responsible for human brain malformation syndrome (holoprosencephaly, or HPE). In embryonic stages, Zic5 was expressed in dorsal part of neural tissues and limbs. Expression of Zic5 overlapped with those of other Zic genes, most closely with Zic2 , but was not identical. Targeted disruption of Zic5 resulted in insufficient neural tube closure at the rostral end, similar to that seen in Zic2 mutant mice. In addition, the Zic5 -deficient mice exhibited malformation of neural-crest-derived facial bones, especially the mandible, which had not been observed in other Zic family mutants. During the embryonic stages, there were delays in the development of the first branchial arch and extension of the trigeminal and facial nerves. Neural crest marker staining revealed fewer neural crest cells in the dorsal cephalic region of the mutant embryos without significant changes in their migration. When mouse Zic5 was overexpressed in Xenopus embryos, expression of a neural crest marker was enhanced. These findings suggested that Zic5 is involved in the generation of neural crest tissue in mouse development. ZIC5 is also located close to ZIC2 in humans, and deletions of 13q32, where ZIC2 is located, lead to congenital brain and digit malformations known as the “13q32 deletion syndrome”. Based on both their similar expression pattern in mouse embryos and the malformations observed in Zic5 -deficient mutant mice, human ZIC5 might be involved in the deletion syndrome.

Mouse Zic5 deficiency results in neural tube defects and hypoplasia of cephalic neural crest derivatives

Zic family genes encode zinc finger proteins, which are homologues of the Drosophila pair-rule gene odd-paired. In the present study, we characterized the fifth member of the mouse Zic family gene, mouse Zic5. Zic5 is located near Zic2, which is responsible for human brain malformation syndrome (holoprosencephaly, or HPE). In embryonic stages, Zic5 was expressed in dorsal part of neural tissues and limbs. Expression of Zic5 overlapped with those of other Zic genes, most closely with Zic2, but was not identical. Targeted disruption of Zic5 resulted in insufficient neural tube closure at the rostral end, similar to that seen in Zic2 mutant mice. In addition, the Zic5-deficient mice exhibited malformation of neural-crest-derived facial bones, especially the mandible, which had not been observed in other Zic family mutants. During the embryonic stages, there were delays in the development of the first branchial arch and extension of the trigeminal and facial nerves. Neural crest marker staining revealed fewer neural crest cells in the dorsal cephalic region of the mutant embryos without significant changes in their migration. When mouse Zic5 was overexpressed in Xenopus embryos, expression of a neural crest marker was enhanced. These findings suggested that Zic5 is involved in the generation of neural crest tissue in mouse development. ZIC5 is also located close to ZIC2 in humans, and deletions of 13q32, where ZIC2 is located, lead to congenital brain and digit malformations known as the “13q32 deletion syndrome”. Based on both their similar expression pattern in mouse embryos and the malformations observed in Zic5-deficient mutant mice, human ZIC5 might be involved in the deletion syndrome.

Zic1 Promotes the Expansion of Dorsal Neural Progenitors in Spinal Cord by Inhibiting Neuronal Differentiation

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differentiation is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick.

Molecular Properties of Zic Proteins as Transcriptional Regulators and Their Relationship to GLI Proteins

Zic family genes encode zinc finger proteins, which play important roles in vertebrate development. The zinc finger domains are highly conserved between Zic proteins and show a notable homology to those of Gli family proteins. In this study, we investigated the functional properties of Zic proteins and their relationship to the GLI proteins. We first established an optimal binding sequence for Zic1, Zic2, and Zic3 proteins by electrophoretic mobility shift assay-based target selection and mutational analysis. The selected sequence was almost identical to the GLI binding sequence. However, the binding affinity was lower than that of GLI. Consistent results were obtained in reporter assays, in which transcriptional activation by Zic proteins was less dependent on the GLI binding sequence than GLI1. Moreover, Zic proteins activated a wide range of promoters irrespective of the presence of a GLI binding sequence. When Zic and GLI proteins were cotransfected into cultured cells, Zic proteins enhanced or suppressed sequence-dependent, GLI-mediated transactivation depending on cell type. Taken together, these results suggest that Zic proteins may act as transcriptional coactivators and that their function may be modulated by the GLI proteins and possibly by other cell type-specific cofactors.

Chromosomal, in silico and in vitro Expression Analysis of Cardiovascular-based Genes Encoding Zinc Finger Proteins

Three hundred and sixty expressed sequence tags (ESTs) from human heart cDNA libraries corresponding to one hundred and twenty six unique zinc finger proteins (ZFPs) were annotated and classified into seven types of ZFPs as reported previously. Among these 126 cvbZFPs (cardiovascular-based ZFPs), the C2H2-type and the C2C2-type are the two major ZFP types which account for more than 80% of ZFP genes present in the cardiovascular system. The expression patterns of 11 randomly selected ZFP genes (at least one for each type) in normal fetal, adult and hypertrophic adult hearts, respectively, were determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The results suggest that ZFPs may be involved in the processes of either developmental control (downregulated or upregulated expression) or basic cellular functional regulation (constant expression). Interestingly, PAF-1 (peroxisome assembly factor-1), a C3HC4-type ZFP (RING domain-containing ZFP) showing a downregulated expression pattern in normal tissues was found to be upregulated in hypertrophic adult heart, suggesting a possible role for this fetal gene in the pathogenesis of cardiac hypertrophy. In silico Northern analysis of 15 tissues showed that over 90% of cvbZFPs demonstrate widespread tissue distribution, suggesting the vast majority of ZFPs are functionally shared among tissues. The potential importance of transcriptional repressors in cardiovascular development and disease, such as HFHZ, was supported by the observation that one-third (39 of 126) of cvbZFPs possess this function. Of these, 26 are C2H2-type and the remaining 13 included 8 C2C2-type, 1 C3HC4-type, 1 C2HC4C(HD)-type, 2 C3H-type and 1 combination type. Of particular interest was the observation that ZFPs which contain a KRAB domain are the major subtype present (51.3% of the total repressors in cvbZFPs). Chromosomal distribution analysis showed that mapping loci of cvbZFP genes are concentrated on chromosomes 1, 3, 6, 8, 10, 11, 12, 19 and X. In particular, chromosome 19 appears to be enriched in ZFP genes with C2H2-type as the predominant type present. Overall, this report provides a fundamental initial step toward understanding the potential role of ZFPs in regulating cadiac development and disease.