Familial Immune Thrombocytopenia Associated With a Novel Variant in IKZF1

We report a novel variant in IKZF1 associated with IKAROS haploinsufficiency in a patient with familial immune thrombocytopenia (ITP). IKAROS, encoded by the IKZF1 gene, is a hematopoietic zinc-finger transcription factor that can directly bind to DNA. We show that the identified IKZF1 variant (p.His195Arg) alters a completely conserved histidine residue required for the folding of the third zinc-finger of IKAROS protein, leading to a loss of characteristic immunofluorescence nuclear staining pattern. In our case, genetic testing was essential for the diagnosis of IKAROS haploinsufficiency, of which known presentations include infections, aberrant hematopoiesis, leukemia, and age-related decrease in humoral immunity. Our family study underscores that, after infections, ITP is the second most common clinical manifestation of IKAROS haploinsufficiency

PhD

dissertationProper gene expression relies on the precise coordination of cellular processes that influence packaging, transcription, and processing of the genetic material. Linkage and regulation of these processes is organized by factors that remodel and modify nucleosomes, regulate transcription, and influence RNA processing and export. One of these factors, Spt6, is a large (~168kDa), essential, highly conserved, and functionally diverse eukaryotic protein. Best known as a histone chaperone capable of altering the structure of nucleosomes, Spt6 has also been shown to function as a transcription elongation factor as well as a critical component for proper RNA processing. Although a broader role for Spt6 is reasonably well-understood, very little is known about the functional and mechanistic details of this multifaceted protein. Beyond studying Spt6 directly, insight into Spt6 function may come from complimentary studies on the bacterial protein Tex. Tex is a transcription elongation factor predicted to be a structurally similar to Spt6. The function of Tex is not well-understood, but may be functioning in a homologous manner to Spt6 in two vastly different transcriptional environments. In order to gain insight into the mechanism of Spt6 and Tex, the work presented in this thesis has focused on structural and biochemical studies of Spt6 from Saccharomyces cerevisiae and the related Tex protein from Pseudomonas aeruginosa. To this end, several Spt6 crystal structures have been determined resulting in a nearly complete composite model for Spt6. Along with a series of domains predicted to mediate protein and nucleic acid interactions, the structure reveals a novel tandem SH2 domain consisting of the only two SH2 folds known in yeast. Biochemical analysis of Spt6 demonstrates its capacity to interact with an array of functionally relevant protein and nucleic acid substrates which provide clues into mechanisms underlying the various functions of Spt6. Parallel studies on Tex demonstrate a strikingly similar structure and domain architecture to that of the Spt6 core. Structural and biochemical work described in this thesis lays the foundation for further in vitro and in vivo studies aimed at a better understanding of how Spt6 and Tex regulate gene expression. The highly similar core structure shared between Spt6 and Tex may ultimately prove to be a protein scaffold for regulating transcription in both eukaryotic and prokaryotic organisms

A new family of β-helix proteins with similarities to the polysaccharide lyases

Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacteriumClostridium thermocellum. Here, the crystal structure of Cthe_2159 is presented and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. Importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.</jats:p

Abstract 3907: Emergent properties of EWS/FLI regulation via GGAA-microsatellites in Ewing's sarcoma

Abstract ETS proteins are a family of transcription factors that play important roles in the development of cancer. The Ewing's sarcoma EWS/ETS fusion oncoproteins control a number of cancer-relevant phenotypes in that disease. We recently demonstrated that EWS/FLI, the most common EWS/ETS fusion in Ewing's sarcoma, regulates a portion of its target genes, including the critical target NR0B1, via GGAA-containing microsatellites in their promoters. Given the unusual nature of microsatellites as EWS/FLI response elements, we sought to elucidate the mechanism of EWS/FLI activity at these sites. In addition to EWS/FLI, we found that other Ewing's sarcoma associated EWS/ETS fusions are capable of mediating gene regulation via the NR0B1 microsatellite. Furthermore, regulation via the GGAA microsatellite was specific to Ewing's sarcoma fusion proteins as two other ETS family members, ETS1 and ELF1, could not cause transcriptional activation, highlighting a neomorphic function of the Ewing's sarcoma fusion proteins. The stoichiometry of this interaction is 2 protein molecules for each DNA molecule, suggesting that EWS/FLI binds these elements as a homodimer. The isolated FLI ETS domain bound microsatellite sequences in a nearly identical fashion to full-length EWS/FLI, thus indicating that residues required for homodimeric binding are localized to the ETS domain. These data suggest a new paradigm for ETS family member binding to DNA at cancer-relevant genetic loci, and highlight emergent properties of EWS/FLI that are required for the development of Ewing's sarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3907.</jats:p

Emergent Properties of EWS/FLI Regulation via GGAA Microsatellites in Ewing’s Sarcoma

ETS proteins are a family of transcription factors that play important roles in the development of cancer. The Ewing’s sarcoma EWS/ETS fusion oncoproteins control a number of cancer-relevant phenotypes in that disease. We recently demonstrated that EWS/FLI, the most common EWS/ETS fusion in Ewing’s sarcoma, regulates a portion of its target genes, including the critical target NR0B1, via GGAA-containing microsatellites in their promoters. Given the unusual nature of microsatellites as EWS/FLI response elements, we sought to elucidate the mechanism of EWS/FLI activity at these sites. We found that the ability to bind GGAA microsatellites is shared by multiple ETS family members from distinct phylogenetic subfamilies. Importantly, however, only EWS/ETS-containing fusions are capable of mediating transcriptional activation via these elements, highlighting a neomorphic function of the Ewing’s sarcoma fusion proteins. Additional analysis revealed that the GGAA microsatellite binds EWS/FLI with an affinity that is 2 to 3 orders of magnitude lower than previously identified high-affinity consensus/redundant binding sites. The stoichiometry of this interaction is 2 protein molecules for each DNA molecule, suggesting that EWS/FLI binds these elements as a homodimer. The isolated FLI ETS domain bound microsatellite sequences in a nearly identical fashion to full-length EWS/FLI, thus indicating that residues required for homodimeric binding are localized to the ETS domain. These data suggest a new paradigm for an ETS family member binding to DNA at cancer-relevant genetic loci and highlight emergent properties of EWS/FLI that are required for the development of Ewing’s sarcoma

Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera

Abstract In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.</jats:p