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

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

X-ray crystal structure and properties of phanta, a weakly fluorescent photochromic GFP-like protein

Phanta is a reversibly photoswitching chromoprotein (Phi(F), 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (Phi(F), 0.8). We have investigated the contribution of substitutions at positions His193, Thr69 and Gln62, individually and in combination, to the optical properties of Phanta. Single amino acid substitutions at position 193 resulted in proteins with very low Phi(F), indicating the importance of this position in controlling the fluorescence efficiency of the variant proteins. The substitution Thr69Val in Phanta was important for supressing the formation of a protonated chromophore species observed in some His193 substituted variants, whereas the substitution Gln62Met did not significantly contribute to the useful optical properties of Phanta. X-ray crystal structures for Phanta (2.3 angstrom), eCGP123(T69V) (2.0 angstrom) and eCGP123(H193Q) (2.2 angstrom) in their non-photoswitched state were determined, revealing the presence of a cis-coplanar chromophore. We conclude that changes in the hydrogen-bonding network supporting the cis-chromophore, and its contacts with the surrounding protein matrix, are responsible for the low fluorescence emission of eCGP123 variants containing a His193 substitution

Optical spectra of Phanta and selected variants.

<p>(A) Spectra determined at pH 8.0 are shown for Phanta and variants of eCGP123 containing amino acid substitutions that contribute to Phanta. Absorbance (solid line), fluorescence excitation (dashed line) and fluorescence emission (dotted line) are shown. (B) The absorbance spectra are shown for selected variants at pH 8.0 (solid line), pH 6.0 (dashed line) and pH 3.0 (dotted line). (C) Absorbance spectra determined at pH 8.0 are shown for variants of eCGP123 singly substituted at position 193 or doubly substituted at positions 193 and 69.</p

Phanta structure.

<p>(A) A schematic ribbon representation of an isolated protomer of Phanta (grey) showing two views (one rotated through 90°) of the 11-stranded β-can motif typical of GFP-like proteins and the central α-helix with the chromophore (orange) represented in stick format. (B) Orthogonal views of the final 2<i>Fo-Fc</i> electron density superposed onto the structure for each of the four protomers of Phanta, and protomer A of eCGP123^T69V and eCGP123^H193Q.</p

The chromophore environment of Phanta, eCGP123^T69V, eCGP123^H193Q and eCGP123.

<p>Stereoviews are shown comparing the chromophore environments and hydrogen bond networks for (A) Phanta; (B) eCGP123^T69V; (C) eCGP123^H193Q and (D) eCGP123. Numbered waters are shown as red spheres.</p

Phanta, eCGP123^T69V and eCGP123^H193Q data collection and refinement statistics.

<p>^a Values in parentheses refer to the highest resolution shell.</p><p>^b<i>R</i>free was calculated with 5% of the diffraction data selected randomly and excluded from refinement.</p><p>Phanta, eCGP123^T69V and eCGP123^H193Q data collection and refinement statistics.</p