ChIP-Chip Identifies SEC23A, CFDP1, and NSD1 as TFII-I Target Genes in Human Neural Crest Progenitor Cells.

Objectives : GTF2I and GTF2IRD1 genes located in Williams-Beuren syndrome (WBS) critical region encode TFII-I family transcription factors. The aim of this study was to map genomic sites bound by these proteins across promoter regions of developmental regulators associated with craniofacial development. Design : Chromatin was isolated from human neural crest progenitor cells and the DNA-binding profile was generated using the human RefSeq tiling promoter ChIP-chip arrays. Results : TFII-I transcription factors are recruited to the promoters of SEC23A, CFDP1, and NSD1 previously defined as TFII-I target genes. Moreover, our analysis revealed additional binding elements that contain E-boxes and initiator-like motifs. Conclusions : Genome-wide promoter binding studies revealed SEC23A, CFDP1, and NSD1 linked to craniofacial or dental development as direct TFII-I targets. Developmental regulation of these genes by TFII-I factors could contribute to the WBS-specific facial dysmorphism.

Diversity and complexity in chromatin recognition by TFII-I transcription factors in pluripotent embryonic stem cells and embryonic tissues.

GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a "feed-forward model" of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells.

Epigenetic modulation by TFII-I during embryonic stem cell differentiation.

TFII-I transcription factors play an essential role during early vertebrate embryogenesis. Genome-wide mapping studies by ChIP-seq and ChIP-chip revealed that TFII-I primes multiple genomic loci in mouse embryonic stem cells and embryonic tissues. Moreover, many TFII-I-bound regions co-localize with H3K4me3/K27me3 bivalent chromatin within the promoters of lineage-specific genes. This minireview provides a summary of current knowledge regarding the function of TFII-I in epigenetic control of stem cell differentiation.

PI3K/Akt-dependent functions of TFII-I transcription factors in mouse embryonic stem cells.

Activation of PI3K/Akt signaling is sufficient to maintain the pluripotency of mouse embryonic stem cells (mESC) and results in down-regulation of Gtf2i and Gtf2ird1 encoding TFII-I family transcription factors. To investigate how these genes might be involved in the process of embryonic stem cell differentiation, we performed expression microarray profiling of mESC upon inhibition of PI3K by LY294002. This analysis revealed significant alterations in expression of genes for specific subsets of chromatin-modifying enzymes. Surprisingly, genome-wide promoter ChIP-chip mapping indicated that the majority of differently expressed genes could be direct targets of TFII-I regulation. The data support the hypothesis that upregulation of TFII-I factors leads to activation of a specific group of developmental genes during mESC differentiation.

Molecular basis of Williams-Beuren syndrome: TFII-I regulated targets involved in craniofacial development.

OBJECTIVE: The aim of this study is to identify gene targets of TFII-I transcription factors involved in craniofacial development. DESIGN: Recent findings in individuals with Williams-Beuren syndrome who show facial dysmorphism and cognitive defects have pointed to TFII-I genes (GTF2I and GTF2IRD1) as the prime candidates responsible for these clinical features. However, TFII-I proteins are multifunctional transcriptional factors regulating a number of genes during development, and how their haploinsufficiency leads to the Williams-Beuren syndrome phenotype is currently unknown. RESULTS: Here we report the identification of three genes with a well-established relevance to craniofacial development as direct TFII-I targets. These genes, craniofacial development protein 1 (Cfdp1), Sec23 homolog A (Sec23a), and nuclear receptor binding SET domain protein 1 (Nsd1), contain consensus TFII-I binding sites in their proximal promoters; the chromatin immunoprecipitation analysis showed that TFII-I transcription factors are recruited to these sites in vivo. CONCLUSIONS: The results suggest that transcriptional regulation of these genes by TFII-I proteins could provide a possible genotype-phenotype link in Williams-Beuren syndrome.

New TFII-I family target genes involved in embryonic development.

Two members of the TFII-I family transcription factor genes, GTF2I and GTF2IRD1, are the prime candidates responsible for the craniofacial and cognitive abnormalities of Williams syndrome patients. We have previously generated mouse lines with targeted disruption of Gtf2i and Gtf2ird1. Microarray analysis revealed significant changes in the expression profile of mutant embryos. Here we described three unknown genes that were dramatically down-regulated in mutants. The 2410018M08Rik/Scand3 gene encodes a protein of unknown function with CHCH and hATC domains. Scand3 is down-regulated during mouse embryonic stem cell (ES) differentiation. 4933436H12Rik is a testis-specific gene, which encodes a protein with no known domains. It is expressed in mouse ES cells. 1110008P08Rik/Kbtbd7 encodes an adapter protein with BTB/POZ, BACK, and Kelch motifs, previously shown to recruit substrates to the enzymatic complexes of the histone modifying or E3 ubiquitin ligase activities. Based on its expression pattern Kbtbd7 may have a specific role in brain development and function. All three genes possess well-conserved TFII-I-binding consensus sites within proximal promoters. Therefore our analysis suggests that these genes can be direct targets of TFII-I proteins and their impaired expression, as a result of the GTF2I and GTF2IRD1 haploinsufficiency, could contribute to the etiology of Williams syndrome.

Alternative splicing and promoter use in TFII-I genes.

TFII-I proteins are ubiquitously expressed transcriptional factors involved in both basal transcription and signal transduction activation or repression. TFII-I proteins are detected as early as at two-cell stage and exhibit distinct and dynamic expression patterns in developing embryos as well as mark regional variation in the adult mouse brain. Analysis of atypical small and rare chromosomal deletions at 7q11.23 points to TFII-I genes (GTF2I and GTF2IRD1) as the prime candidates responsible for craniofacial and cognitive abnormalities in the Williams-Beuren syndrome. TFII-I genes are often subjected to alternative splicing, which generates isoforms that show different activities and play distinct biological roles. The coding regions of TFII-I genes are composed of more than 30 exons and are well conserved among vertebrates. However, their 5' untranslated regions are not as well conserved and all poorly characterized. In the present work, we analyzed promoter regions of TFII-I genes and described their additional exons, as well as tested tissue specificity of both previously reported and novel alternatively spliced isoforms. Our comprehensive analysis leads to further elucidation of the functional heterogeneity of TFII-I proteins, provides hints on search for regulatory pathways governing their expression, and opens up possibilities for examining the effect of different haplotypes on their promoter functions.

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.

Identification of the TFII-I family target genes in the vertebrate genome.

GTF2I and GTF2IRD1 encode members of the TFII-I transcription factor family and are prime candidates in the Williams syndrome, a complex neurodevelopmental disorder. Our previous expression microarray studies implicated TFII-I proteins in the regulation of a number of genes critical in various aspects of cell physiology. Here, we combined bioinformatics and microarray results to identify TFII-I downstream targets in the vertebrate genome. These results were validated by chromatin immunoprecipitation and siRNA analysis. The collected evidence revealed the complexity of TFII-I-mediated processes that involve distinct regulatory networks. Altogether, these results lead to a better understanding of specific molecular events, some of which may be responsible for the Williams syndrome phenotype.

HnRNP A3 genes and pseudogenes in the vertebrate genomes.

The hnRNP A/B type proteins are abundant nuclear factors that bind to Pol II transcripts and are involved in numerous RNA-related activities. To date most data on the hnRNP A/B family have been obtained with recombinant proteins and cell cultures. Further characterization can result from an examination of the impact of various modifications in intact functional loci; however, such characterization is hampered by the presence of numerous and widely dispersed hnRNP A/B-related sequences in the mammalian genome. We have found hnRNP A3, a poorly recognized member of the hnRNP A/B family, among candidate transcription factors that interact with the regulatory region of the Hoxc8 gene and screened the human and mouse genomes for genes that encode hnRNP A3. We demonstrate that the sequence reported previously as the human hnRNP A3 gene (Accession number S63912) and located on 10p11.1 belongs to a processed pseudogene of the functional intron-containing locus HNRPA3, which we have identified on 2q31.2. We have also identified its murine orthologs on mouse chromosome 2D and rat chromosome 3q23. Alternative splices were revealed at the N-terminus and in the middle of hnRNP A3. 14 and 28 additional loci in the human and mouse genome, respectively, were mapped and identified as hnRNP A3 processed pseudogenes. In addition, we have found and compared hnRNP A3 orthologous genes in Gallus gallus, Xenopus tropicalis, and Danio rerio. The present in silico analysis serves as a necessary step toward a further functional characterization of hnRNP A3.

GTF2IRD2 is located in the Williams-Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix-loop-helix repeats.

Williams-Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix-loop-helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix-loop-helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams-Beuren syndrome critical region.

Targeting a KH-domain protein with RNA decoys.

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.

The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms.

The poly(C) binding proteins (PCBPs) are encoded at five dispersed loci in the mouse and human genomes. These proteins, which can be divided into two groups, hnRNPs K/J and the alphaCPs (alphaCP1-4), are linked by a common evolutionary history, a shared triple KH domain configuration, and by their poly(C) binding specificity. Given these conserved characteristics it is remarkable to find a substantial diversity in PCBP functions. The roles of these proteins in mRNA stabilization, translational activation, and translational silencing suggest a complex and diverse set of post-transcriptional control pathways. Their additional putative functions in transcriptional control and as structural components of important DNA-protein complexes further support their remarkable structural and functional versatility. Clearly the identification of additional binding targets and delineation of corresponding control mechanisms and effector pathways will establish highly informative models for further exploration.