Aleksandr V. Makeyev

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 TGFβRII/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.

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.

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.

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. J. Cell. Biochem. 113: 1122–1131, 2012.

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. J. Cell. Biochem. 113: 3056–3060, 2012.

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.

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.