Paola Izzo

KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions

Zinc finger proteins containing the Kruppel associated box (KRAB-ZFPs) constitute the largest individual family of transcriptional repressors encoded by the genomes of higher organisms. KRAB domain, positioned at the NH2 terminus of the KRAB-ZFPs, interacts with a scaffold protein, KAP-1, which is able to recruit various transcriptional factors causing repression of genes to which KRAB ZFPs bind. The relevance of such repression is reflected in the large number of the KRAB zinc finger protein genes in the human genome. However, in spite of their numerical abundance little is currently known about the gene targets and the physiological functions of KRAB- ZFPs. However, emerging evidence links the transcriptional repression mediated by the KRAB-ZFPs to cell proliferation, differentiation, apoptosis and cancer. Moreover, the fact that KRAB containing proteins are vertebrate-specific suggests that they have evolved recently, and that their key roles lie in some aspects of vertebrate development. In this review, we will briefly discuss some regulatory functions of the KRAB-ZFPs in different physiological and pathological states, thus contributing to better understand their biological roles.

Effects of the R216Q mutation of GATA-1 on erythropoiesis and megakaryocytopoiesis

The transcription factor GATA-1, together with its cofactor FOG-1, regulates erythropoiesis and megakaryocytopoiesis. Mutations in the DNA or FOG-1 binding sites of its N-terminal zinc finger result in different illnesses. Alterations of the FOG-1 face are responsible for dyserythropoietic anemia with thrombocytopenia while R216Q, the only mutation identified in the DNA face, induces X-linked thrombocytopenia with thalassemia (XLTT). The former disorder has been studied in detail whereas little is known about the latter since only one family has been investigated. We studied a second family with an R216Q, showing that XLTT and dyserythropoietic anemia with thrombocytopenia, even if different clinical entities, are closely related disorders. In both cases, patients present mild dyserythropoiesis, red cell hemolysis, severely defective maturation of megakaryocytes, macrothrombocytopenia with alpha-granule deficiency, and abnormalities of the cytoplasmic membrane system. However, a thalassemia minor phenotype has only been described in patients with XLTT whereas severe anemia and thrombocytopenia with evident defects of platelet composition and function may be observed only in dyserythropoietic anemia with thrombocytopenia.

Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial‐derived S100B protein

Abstract  In the central nervous system glial‐derived S100B protein has been associated with inflammation via nitric oxide (NO) production. As the role of enteroglial cells in inflammatory bowel disease has been poorly investigated in humans, we evaluated the association of S100B and NO production in ulcerative colitis (UC). S100B mRNA and protein expression, inducible NO synthase (iNOS) expression, and NO production were evaluated in rectal biopsies from 30 controls and 35 UC patients. To verify the correlation between S100B and NO production, biopsies were exposed to S100B, in the presence or absence of specific receptor for advanced glycation end‐products (RAGE) blocking antibody, to measure iNOS expression and nitrite production. S100B and iNOS expression were evaluated after incubation of biopsies with lipopolysaccharides (LPS) + interferon‐gamma (IFN‐γ) in the presence of anti‐RAGE or anti‐S100B antibodies or budesonide. S100B mRNA and protein expression, iNOS expression and NO production were significantly higher in the rectal mucosa of patients compared to that of controls. Exogenous S100B induced a significant increase in both iNOS expression and NO production in controls and UC patients; this increase was inhibited by specific anti‐RAGE blocking antibody. Incubation with LPS + IFN‐γ induced a significant increase in S100B mRNA and protein expression, together with increased iNOS expression and NO production. LPS + IFN‐γ‐induced S100B up‐regulation was not affected by budesonide, while iNOS expression and NO production were significantly inhibited by both specific anti‐RAGE and anti‐S100B blocking antibodies. Enteroglial‐derived S100B up‐regulation in UC participates in NO production, involving RAGE in a steroid insensitive pathway.

Genetics, diagnosis and management of colorectal cancer (Review)

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. Surgery represents the mainstay of treatment in early cases but often patients are primarily diagnosed in an advanced stage of disease and sometimes also distant metastases are present. Neoadjuvant therapy is therefore needed but drug resistance may influence response and concur to recurrent disease. At molecular level, it is a very heterogeneous group of diseases with about 30% of hereditary or familial cases. During colorectal adenocarcinomas development, epithelial cells from gastrointestinal trait acquire sequential genetic and epigenetic mutations in specific oncogenes and/or tumour suppressor genes, causing CRC onset, progression and metastasis. Molecular characterization of cancer associated mutations gives valuable information about disease prognosis and response to the therapy. Very early diagnosis and personalized care, as well as a better knowledge of molecular basis of its onset and progression, are therefore crucial to obtain a cure of CRC. In this review, we describe updated genetics, current diagnosis and management of CRC pointing out the extreme need for a multidisciplinary approach to achieve the best results in patient outcomes.

Three submicroscopic deletions at the APC locus and their rapid detection by quantitative-PCR analysis.

We describe three unrelated kindreds, affected by familial adenomatous polyposis (FAP), with 5q submicroscopic deletions that encompass the entire adenomatous polyposis coli (APC) gene and the adjacent DP1 gene. In one family the deletion encompasses also the MCC (mutated in colon cancer) gene. Affected members of these families had dysplastic adenomatous polyps and congenital hypertrophy of the retinal pigment epithelium (CHRPE); no individual was affected by mental retardation or facial dysmorphism. The deletions were detected by linkage analysis with several intragenic and closely flanking polymorphic markers and confirmed by a quantitative PCR analysis. This procedure could have an impact on the detection of the molecular defect in FAP patients in whom mutational analysis fails to identify the specific mutation.

The Kruppel-like Zinc Finger Protein ZNF224 Recruits the Arginine Methyltransferase PRMT5 on the Transcriptional Repressor Complex of the Aldolase A Gene

Gene transcription in eukaryotes is modulated by the coordinated recruitment of specific transcription factors and chromatin-modulating proteins. Indeed, gene activation and/or repression is/are regulated by histone methylation status at specific arginine or lysine residues. In this work, by co-immunoprecipitation experiments, we demonstrate that PRMT5, a type II protein arginine methyltransferase that monomethylates and symmetrically dimethylates arginine residues, is physically associated with the Kruppel-like associated box-zinc finger protein ZNF224, the aldolase A gene repressor. Moreover, chromatin immunoprecipitation assays show that PRMT5 is recruited to the L-type aldolase A promoter and that methylation of the nucleosomes that surround the L-type promoter region occurs in vivo on the arginine 3 of histone H4. Consistent with its association to the ZNF224 repressor complex, the decrease of PRMT5 expression produced by RNA interference positively affects L-type aldolase A promoter transcription. Finally, the alternating occupancy of the L-type aldolase A promoter by the ZNF224-PRMT5 repression complex in proliferating and growth-arrested cells suggests that these regulatory proteins play a significant role during the cell cycle modulation of human aldolase A gene expression. Our data represent the first experimental evidence that protein arginine methylation plays a role in ZNF224-mediated transcriptional repression and provide novel insight into the chromatin modifications required for repression of gene transcription by Kruppel-like associated box-zinc finger proteins.

WT1 protein is a transcriptional activator of the antiapoptotic bag3 gene

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A novel zinc finger transcriptional repressor, ZNF224, interacts with the negative regulatory element (AldA-NRE) and inhibits gene expression.

The interaction between the negative cis‐element (AldA‐NRE) and p97 repressor nuclear protein is a key step in modulating transcription of the human and mouse aldolase A (AldA) gene during the cell cycle and differentiation. In an attempt to clarify the role of transcriptional repression in regulating gene expression, we purified, from HeLa cells, the nuclear protein that specifically binds to the AldA negative regulatory element (NRE). Matrix‐assisted laser desorption ionization‐time of flight analysis and examination of protein profiles from the SwissProt database revealed that the previously defined p97 repressor is ZNF224, a zinc finger protein. We demonstrate that ZNF224, a Kruppel‐like zinc finger transcription factor, is the repressor protein that specifically binds to the negative cis‐element AldA‐NRE and affects the AldA‐NRE‐mediated transcription.

Association of low‐risk MSH3 and MSH2 variant alleles with Lynch syndrome: Probability of synergistic effects

Mutations in the MLH1 and MSH2 genes account for a majority of cases of families with Lynch Syndrome. Germ‐line mutations in MSH6, PMS2 and MLH3 are responsible for disease in a minority of cases, usually associated with milder and variable phenotypes. No germ‐line mutations in MSH3 have so far been associated with Lynch Syndrome, although it is known that impaired MSH3 activity leads to a partial defect in mismatch repair (MMR), with low levels of microsatellite instability at the loci with dinucleotide repeats in colorectal cancer (CRC), thus suggesting a role for MSH3 in carcinogenesis. To determine a possible role of MSH3 as predisposing to CRC in Lynch syndrome, we screened MSH3 for germ‐line mutations in 79 unrelated Lynch patients who were negative for pathogenetic mutations in MLH1, MSH2 and MSH6. We found 13 mutant alleles, including silent, missense and intronic variants. These variants were identified through denaturing high performance liquid chromatography and subsequent DNA sequencing. In one Lynch family, the index case with early‐onset colon cancer was a carrier of a polymorphism in the MSH2 gene and two variants in the MSH3 gene. These variants were associated with the disease in the family, thus suggesting the involvement of MSH3 in colon tumour progression. We hypothesise a model in which variants of the MSH3 gene behave as low‐risk alleles that contribute to the risk of colon cancer in Lynch families, mostly with other low‐risk alleles of MMR genes.

Wnt pathway, angiogenetic and hormonal markers in sporadic and familial adenomatous polyposis-associated juvenile nasopharyngeal angiofibromas (JNA)

Juvenile nasopharyngeal angiofibroma (JNA) is a rare, invasive, and locally destructive tumor of the nasopharynx. The Wnt pathway, angiogenetic and hormonal factors are involved in the pathophysiology of JNA; it can result in an extracolonic manifestation of familial adenomatous polyposis (FAP) or in a sporadic tumor. All patients who underwent resection of JNA between 1991 and 2006 at the University of Modena and Reggio Emilia were studied to identify immunohistochemical markers of associated FAP syndrome. Paraffin-embedded JNA samples were analyzed immunohistochemically for the expression of adenomatous polyposis coli (APC), β-catenin, E-cadherin, androgen receptor, and vascular endothelial growth factors receptor (VEGFR2). In one out of the 4 (25%) young patients affected by JNA the diagnosis of FAP syndrome linked to APC mutation was made. All of the sporadic and familial JNA tumors showed nuclear staining of β-catenin, whereas altered APC expression was seen only in FAP-associated JNA. All cases were stained with VEGFR2. A combined clinical, immunohistochemical, and biomolecular screening may be useful for the identification of FAP among patients with a diagnosis of JNA. The Wnt pathway can be involved in the JNA pathogenesis either by somatic mutations of β-catenin or by germline APC mutations. As the VEGFR has an important impact on the pathogenesis of JNA, we suggest that a targeted therapy with monoclonal antibodies against VEGFR might lead to a specific chemoprevention and treatment of these tumors and their recurrences.