Protein and gene expression analysis of Phf6, the gene mutated in the Börjeson–Forssman–Lehmann Syndrome of intellectual disability and obesity

Structural and Functional Insights into the Human Börjeson-Forssman-Lehmann Syndrome-associated Protein PHF6

“…15 N-or 15 N/ 13 C-labeled proteins were produced in SV40 medium supplemented with 15 NH 4 Cl or 15 NH 4 Cl and [ 13 C 6 ]glucose. The fusion protein was purified on glutathione-Sepharose beads (GE Healthcare).…”

“…All NMR data were processed using the NMRPipe (34) and analyzed by Sparky (T. D. Goddard and D. G. Kneller, University of California at San Francisco). 15 N relaxation measurements were performed on a Bruker DMX-600 equipped with a cryoprobe using a 15 N-labeled PHF6-ePHD2 sample with a concentration of 0.6 mM at 298 K. The 1 H-15 N heteronuclear NOE experiments were recorded in an interleaved fashion, alternately with and without proton presaturation in the recovery delay. A 2-s relaxation delay following a 3-s 1 H presaturation delay were used in the NOE experiment, and a 5-s relaxation delay was used in the reference experiment.…”

“…Apparent J NH constants of 15 Nlabeled PHF6(208-333) were measured at Agilent 700-MHz spectrometer using the in-phase and anti-phase scheme (36). RDC values were obtained by subtracting the reference value of the protein in isotropic solution.…”

“…No interaction was detected between calf thymus histones and the PHF6-ePHD2 domain in high salt (1 M NaCl) buffer using an in vitro GST pulldown assay (data not shown). NMR titration experiments were also performed to explore the interaction of PHF6-ePHD2 with histone H3 and H4 N-terminal peptides; however, no obvious chemical shift perturbation was observed in 15 N-HSQC spectra (data not shown), implying that PHF6-ePHD2 does not bind histones. Based on the structural information of PHF6-ePHD2, we could give the explanation for why it does not bind to histones.…”

“…PHF6 is highly conserved among vertebrate species, including Mus musculus and Xenopus laevis, but it has no obvious orthologs in nonvertebrates, suggesting the conservative functions of PHF6 in the cell (1). Northern blot and in situ hybridization analyses have revealed that PHF6 is highly expressed during embryonic, fetal, and postnatal stages of brain development and predominantly present in the developing embryonic central nervous system (1,15). Therefore, it appears that PHF6 plays an important role in human brain development.…”