Prevalence of germline mutations in the TP53 gene in patients with early-onset breast cancer in the Mexican population.

BACKGROUND: Heterozygous germline TP53 gene mutations result in Li-Fraumeni Syndrome (LFS). Breast cancer (BC) is the most frequent tumor in young women with LFS. An important issue related to BC in the Mexican population is the average age at diagnosis, which is approximately 11 years younger than that of patients in the United States (U.S.) and Europe. The aim of this study was to determine the prevalence of germline mutations in TP53 among young Mexican BC patients. METHODS: We searched for germline mutations in the TP53 gene using targeted next-generation sequencing (NGS) in 78 BC patients younger than 45 years old (yo) who tested negative for BRCA1/2 mutations. A group of 509 Mexican women aged 45yo or older without personal or family BC history (parents/grandparents) was used as a control. RESULTS: We identified five patients with pathogenic variants in the TP53 gene, equivalent to 6.4% (5/78). Among patients diagnosed at age 36 or younger, 9.4% (5/55) had pathogenic TP53 mutations. Three of these variants were missense mutations (c.844C > T, c.517G > A, and c.604C > T), and the other two mutations were frameshifts (c.291delC and c.273dupC) and had not been reported previously. We also identified a variant of uncertain clinical significance (VUS), c.672G > A, which causes a putative splice donor site mutation. All patients with TP53 mutations had high-grade and HER2-positive tumors. None of the 509 patients in the healthy control group had mutations in TP53. CONCLUSIONS: Among Mexican BC patients diagnosed at a young age, we identified a high proportion with germline mutations in the TP53 gene. All patients with the TP53 mutations had a family history suggestive of LFS. To establish the clinical significance of the VUS found, additional studies are needed. Pathogenic variants of TP53 may explain a substantial fraction of BC in young women in the Mexican population. Importantly, none of these mutations or other pathological variants in TP53 were found in the healthy control group.

A Human Lin- CD123+ CD127low Population Endowed with ILC Features and Migratory Capabilities Contributes to Immunopathological Hallmarks of Psoriasis.

Innate lymphoid cells (ILC) are members of a heterogeneous family with a lymphoid origin that mimics the T helper (Th) cytokine profile. ILC are involved in early effector cytokine-mediated responses during infections in peripheral tissues. ILC also play an important role in chronic skin inflammatory diseases, including psoriasis. Although classical ILC express CD127, it has been recently reported that the presence of non-classical CD127- ILC populations and an early ILC precursor (EILP) CD127low. ILC development has predominately been investigated in mouse models. However, in humans, different transcription factors have been described for ILC identification. NFIL3 (nuclear factor, IL-3 regulated) is crucial for ILC development in response to IL-7. CD123 (IL-3Rα) is usually used to exclude basophils during ILC identification, however, it is unknown if in response to IL-3, NFIL3 could be relevant to induce ILC features in Lin- CD123+ populations in addition, is also unknown whether peripheral blood (PB) population with ILC features may have skin-homing potential to participate in skin inflammatory chronic diseases. Here, we report a Lin- CD123+ CD127low CD7+ CLA+ population that share some phenotypic properties with basophils, but expresses several transcription factors for ILC commitment such as inhibitor of DNA binding 2 (Id2), NFIL3, promyelocytic leukemia zinc finger (PLZF), thymocyte selection-associated high-mobility group box protein (TOX), and T cell factor-1 (TCF-1). In addition, this population expresses different ILC markers: CD132, CD90, CD161, α4 integrin, c-Kit, CRTH2, AhR, and IL-23R. IL-3 prevents apoptosis and increases their NFIL3, TOX, and PLZF expression. In PB, the CD123+ CD127low population is predominantly a conspicuous population that expresses T-bet and RORγt. The Lin- CD123+ CD127low population in PB has a limited Th type cytokine expression and highly expresses IL-8. The Lin- CD123+ CD127low population expresses skin-homing receptors (cutaneous lymphocyte antigen and CXCR4) and transmigrates through endothelial cells in response to SDF-1. An equivalent Lin- CD123low population was identified in control skin, which shows a broader phenotypic diversity and cytokine production, including IL-22 and IL-17. Remarkably, the CD123low population in the lesion and non-lesion skin of psoriasis patients expresses IL-17 and IL-22. Our findings suggest the identification of an alternative Lin- CD123+ CD127low population with ILC features endowed with migratory capabilities that might contribute to immunopathological hallmarks of psoriasis.

Williams-Beuren syndrome-associated transcription factor TFII-I regulates osteogenic marker genes.

Williams-Beuren syndrome (WBS), an autosomal dominant genetic disorder, is characterized by a unique cognitive profile and craniofacial defects. WBS results from a microdeletion at the chromosomal location 7q11.23 that encompasses the genes encoding the members of TFII-I family of transcription factors. Given that the haploinsufficiency for TFII-I is causative to the craniofacial phenotype in humans, we set out to analyze the effect of post-transcriptional silencing of TFII-I during BMP-2-driven osteoblast differentiation in the C2C12 cell line. Our results show that TFII-I plays an inhibitory role in regulating genes that are essential in osteogenesis and intersects with the bone-specific transcription factor Runx2 and the retinoblastoma protein, pRb. Identification of pathways regulated by TFII-I family transcription factors may begin to shed light on the molecular determinants of WBS.

Essential functions of the Williams-Beuren syndrome-associated TFII-I genes in embryonic development.

GTF2I and GTF2IRD1 encoding the multifunctional transcription factors TFII-I and BEN are clustered at the 7q11.23 region hemizygously deleted in Williams-Beuren syndrome (WBS), a complex multisystemic neurodevelopmental disorder. Although the biochemical properties of TFII-I family transcription factors have been studied in depth, little is known about the specialized contributions of these factors in pathways required for proper embryonic development. Here, we show that homozygous loss of either Gtf2ird1 or Gtf2i function results in multiple phenotypic manifestations, including embryonic lethality; brain hemorrhage; and vasculogenic, craniofacial, and neural tube defects in mice. Further analyses suggest that embryonic lethality may be attributable to defects in yolk sac vasculogenesis and angiogenesis. Microarray data indicate that the Gtf2ird1 homozygous phenotype is mainly caused by an impairment of the genes involved in the TGFbetaRII/Alk1/Smad5 signal transduction pathway. The effect of Gtf2i inactivation on this pathway is less prominent, but downregulation of the endothelial growth factor receptor-2 gene, resulting in the deterioration of vascular signaling, most likely exacerbates the severity of the Gtf2i mutant phenotype. A subset of Gtf2ird1 and Gtf2i heterozygotes displayed microcephaly, retarded growth, and skeletal and craniofacial defects, therefore showing that haploinsufficiency of TFII-I proteins causes various developmental anomalies that are often associated with WBS.

Determination and functional analysis of the consensus binding site for TFII-I family member BEN, implicated in Williams-Beuren syndrome.

The ubiquitously expressed TFII-I family of multifunctional transcription factors is involved in gene regulation as well as signaling. Despite the fact that they share significant sequence homology, these factors exhibit varied and distinct functions. The lack of knowledge about its binding sites and physiological target genes makes it more difficult to assign a definitive function for the TFII-I-related protein, BEN. We set out to determine its optimal binding site with the notion of predicting its physiological target genes. Here we report the identification of an optimal binding sequence for BEN by SELEX (systematic evolution of ligands by exponential enrichment) and confirm the relevance of this sequence by functional assays. We further performed a data base search to assign genes that have this consensus site(s) and validate several candidate genes by quantitative PCR upon stable silencing of BEN and by chromatin immunoprecipitation assay upon stable expression of BEN. Given that haploinsufficiency in BEN is causative to Williams-Beuren syndrome, these results may further lead to the identification of a set of physiologically relevant target genes for BEN and may help identify molecular determinants of Williams-Beuren syndrome.

Opposing functions of TFII-I spliced isoforms in growth factor-induced gene expression.

Multifunctional transcription factor TFII-I has two spliced isoforms (Delta and beta) in murine fibroblasts. Here we show that these isoforms have distinct subcellular localization and mutually exclusive transcription functions in the context of growth factor signaling. In the absence of signaling, TFII-Ibeta is nuclear and recruited to the c-fos promoter in vivo. But upon growth factor stimulation, the promoter recruitment is abolished and it is exported out of the nucleus. Moreover, isoform-specific silencing of TFII-Ibeta results in transcriptional activation of the c-fos gene. In contrast, TFII-IDelta is largely cytoplasmic in the resting state but translocates to the nucleus upon growth factor signaling, undergoes signal-induced recruitment to the same site on the c-fos promoter, and activates the gene. Importantly, activated TFII-IDelta interacts with Erk1/2 (MAPK) kinase in the cell cytoplasm and imports the Erk1/2 to the nucleus, thereby transducing growth factor signaling. Our results identify a unique growth factor signaling pathway controlled by opposing activities of two TFII-I spliced isoforms.

Positive and negative regulation of the transforming growth factor beta/activin target gene goosecoid by the TFII-I family of transcription factors.

Goosecoid (Gsc) is a homeodomain-containing transcription factor present in a wide variety of vertebrate species and known to regulate formation and patterning of embryos. Here we show that in embryonic carcinoma P19 cells, the transcription factor TFII-I forms a complex with Smad2 upon transforming growth factor beta (TGFbeta)/activin stimulation, is recruited to the distal element (DE) of the Gsc promoter, and activates Gsc transcription. Downregulation of endogenous TFII-I by small inhibitory RNA in P19 cells abolishes the TGFbeta-mediated induction of Gsc. Similarly, Xenopus embryos with endogenous TFII-I expression downregulated by injection of TFII-I-specific antisense oligonucleotides exhibit decreased Gsc expression. Unlike TFII-I, the related factor BEN (binding factor for early enhancer) is constitutively recruited to the distal element in the absence of TGFbeta/activin signaling and is replaced by the TFII-I/Smad2 complex upon TGFbeta/activin stimulation. Overexpression of BEN in P19 cells represses the TGFbeta-mediated transcriptional activation of Gsc. These results suggest a model in which TFII-I family proteins have opposing effects in the regulation of the Gsc gene in response to a TGFbeta/activin signal.

Regulation of immunoglobulin promoter activity by TFII-I class transcription factors.

The restriction of immunoglobulin variable region promoter activity to B lymphocytes is a well known paradigm of promoter specificity. Recently, a cis-element, located downstream of the transcription initiation site of murine heavy chain variable promoters, was shown to be critical for B cell activity and specificity. Here we show that mutation of this element, termed DICE (Downstream Immunoglobulin Control Element), reduces in vivo activity in B cells. Gel mobility shift assays show that DICE forms B cell-specific complexes that were also sensitive to DICE mutation. DICE mutation strongly reduces the ability of a distal immunoglobulin heavy chain intronic enhancer to stimulate transcription. We also identify a DICE-interacting factor: a TFII-I-related protein known as BEN (also termed Mus-TRD1 and WBSCR11). Dominant-negative and RNAi-mediated knockdown experiments indicate that BEN can both positively and negatively regulate IgH promoter activity, depending on the cell line.

Mechanism of Bruton's tyrosine kinase-mediated recruitment and regulation of TFII-I.

TFII-I is a ubiquitously expressed multifunctional transcription factor with broad biological roles in transcription and signal transduction in a variety of cell types. We and others have shown that TFII-I can interact physically and functionally with Bruton's tyrosine kinase (Btk), a hematopoietic non-receptor protein tyrosine kinase that is critical for B lymphocyte development. Although TFII-I-Btk interactions are impaired in B cells from X-linked immunodeficient mice, the precise molecular determinants governing TFII-I-Btk complex formation remain unknown. To this end, we have conducted a structural analysis of TFII-I-Btk interactions by using a panel of TFII-I mutants. These studies have revealed that a region within the N-terminal 90 amino acids of TFII-I, which includes a putative leucine zipper motif, is primarily responsible for its interaction with Btk. Mutations in the leucine zipper region itself were not sufficient to abrogate binding of TFII-I to Btk, suggesting that regions/residues outside the leucine zipper are responsible for such interactions. Because the first 90 amino acids of TFII-I are required for its dimerization, we propose that Btk tethers TFII-I to the cytoplasm by preventing its dimerization and its subsequent nuclear localization. We further examined the requirement of tyrosine phosphorylation for TFII-I-Btk complex formation. Our data showed that Src-dependent tyrosine phosphorylation sites in TFII-I are not targeted by Btk, suggesting that multiple kinases can independently target TFII-I via distinct signaling pathways. Our results provide a beginning step toward understanding the functional importance of the TFII-I-Btk pathway in B cell signaling and gene expression.

Expression of BEN, a member of TFII-I family of transcription factors, during mouse pre- and postimplantation development.

BEN is a member of the TFII-I family of transcription factors, characterized by the presence of multiple helix-loop-helix repeat domains. Our immunohistochemical analysis demonstrated broad and extensive expression of BEN during mouse pre- and postimplantation development, with highest levels occurring during early to midgestation. Maternally expressed BEN is present in both the cytoplasm and nuclei of the zygote; however, it retains a predominantly nuclear localization between the two-cell stage of development and early blastocyst stages. This nuclear expression is observed in most tissues throughout development. Although, it is interesting to note that at E4.5-6.5, during early gastrulation stage, BEN is localized in the cytoplasm. At later stages, BEN retains an extensive expression pattern in a variety of developing systems implicating its involvement in tissue development and organogenesis.

Physical and functional interactions of histone deacetylase 3 with TFII-I family proteins and PIASxbeta.

TFII-I family proteins are characterized structurally by the presence of multiple reiterated I-repeats, each containing a putative helix-loop-helix domain. Functionally, they behave as multifunctional transcription factors that are activated by a variety of extracellular signals. In studying their subcellular localization, we noticed that these transcription factors frequently reside in subnuclear domains/dots. Because nuclear dots are believed often to harbor components of histone deacetylase enzymes (HDACs), we investigated whether TFII-I family proteins colocalize and interact with HDACs. Here, we show that TFII-I and its related member hMusTRD1/BEN physically and functionally interact with HDAC3. The TFII-I family proteins and HDAC3 also show nearly identical expression patterns in early mouse development. Consistent with our earlier observation that TFII-I family proteins also interact with PIASxbeta, a member of the E3 ligase family involved in the small ubiquitin-like modifier (SUMO) pathway, we show further that PIASxbeta physically and functionally interacts with HDAC3 and relieves the transcriptional repression exerted by HDAC3 upon TFII-I-mediated gene activation. These results suggest a complex interplay between two posttranslational pathways-histone modification and SUMOylation-brokered in part by TFII-I family proteins.

The SUMO ubiquitin-protein isopeptide ligase family member Miz1/PIASxbeta /Siz2 is a transcriptional cofactor for TFII-I.

We have shown previously that a TFII-I-related protein, hMusTRD1/BEN, represses transcriptional activity of TFII-I. The repression by hMusTRD1/BEN was hypothesized to occur via a two-step competition mechanism involving a cytoplasmic shuttling factor and a nuclear cofactor required for transcriptional activation of TFII-I. Employing a two-hybrid approach with both yeast genomic and mouse cDNA libraries in parallel, we have identified the RING-like zinc finger containing Miz1/PIASxbeta/Siz2, which is a ubiquitin-protein isopeptide ligase in the SUMO pathway, as the potential nuclear cofactor that interacts with both TFII-I and hMusTRD1/BEN. Our conclusion is based on the following observations. First, the interactions are biochemically confirmed in mammalian cells where Miz1/mPIASxbeta interacts with both TFII-I and hMusTRD1/BEN when these proteins are ectopically co-expressed. Second, co-expression of a nuclear localization signal-deficient mutant of Miz1/mPIASxbeta with wild type TFII-I fails to alter the subcellular localization of the former. Finally, ectopically expressed Miz1/mPIASxbeta augments the transcriptional activity of TFII-I and relieves the repression exerted by a mutant hMusTRD1/BEN that co-localized with TFII-I in the nucleus.