Interaction between PHOX2B and CREBBP mediates synergistic activation: Mechanistic implications of PHOX2B mutants

Congenital central hypoventilation syndrome (CCHS) is a disorder of the autonomic nervous system. The genetic defect is usually caused by heterozygous mutations of the PHOX2B gene such as a 20‐alanine tract (+5 to+13 alanines) expansion (∼95%) and occasional frameshift or missense mutations. Cytoplasmic aggregates were shown in PHOX2B proteins with longer alanine tract (+9 and longer) expansion and impaired DNA binding was observed in PHOX2B proteins with frameshift, missense, or longer alanine tract (+9 and longer) expansion. Defective transactivation activity was shown in certain PHOX2B mutants. However, PHOX2B proteins with short alanine tract (+5 to+7) expansion in the majority of patients (∼75%) did not have cytoplasmic aggregates or DNA binding defects. CREB‐binding protein (CREBBP/CBP) is a transcriptional coactivator that interacts with multiple transcription factors to cause synergistic activation. Here we show that CBP interacted with PHOX2B and served as its coactivator to mediate synergistic activation. Wild‐type PHOX2B and CBP used specific domains to interact with each other. The domains of CBP that interacted with different PHOX2B mutants were different compared to those interacting with wild‐type PHOX2B. Transient cotransfection assays using different PHOX2B mutants and CBP showed the impaired synergistic activation caused by different PHOX2B mutants. An interfering effect was observed in certain PHOX2B mutants. These results demonstrated that aberrant interaction of PHOX2B mutants with CBP and/or an interfering effect of certain PHOX2B mutants may be the critical mechanism to impair synergistic activation, thereby contributing to the phenotypes of CCHS.Hum Mutat 0, 1–7, 2009. © 2009 Wiley‐Liss, Inc.

[1]  Tomohiko Nakamura,et al.  Molecular analysis of congenital central hypoventilation syndrome , 2003, Human Genetics.

[2]  R. Arkell,et al.  In vitro analysis of partial loss-of-function ZIC2 mutations in holoprosencephaly: alanine tract expansion modulates DNA binding and transactivation. , 2005, Human molecular genetics.

[3]  B. Maher,et al.  Idiopathic congenital central hypoventilation syndrome: Analysis of genes pertinent to early autonomic nervous system embryologic development and identification of mutations in PHOX2b , 2003, American journal of medical genetics. Part A.

[4]  A. Munnich,et al.  Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome , 2003, Nature Genetics.

[5]  David J. Anderson,et al.  Specification of Neurotransmitter Identity by Phox2 Proteins in Neural Crest Stem Cells , 1999, Neuron.

[6]  M. Marazita,et al.  In pursuit (and discovery) of a genetic basis for congenital central hypoventilation syndrome , 2005, Respiratory Physiology & Neurobiology.

[7]  R. Ravazzolo,et al.  Distinct pathogenetic mechanisms for PHOX2B associated polyalanine expansions and frameshift mutations in congenital central hypoventilation syndrome. , 2005, Human molecular genetics.

[8]  Win-Li Lin,et al.  Unequal crossover recombination – population screening for PHOX2B gene polyalanine polymorphism using CE , 2007, Electrophoresis.

[9]  K. Tokunaga,et al.  De novo polyalanine expansion of PHOX2B in congenital central hypoventilation syndrome: unequal sister chromatid exchange during paternal gametogenesis , 2007, Journal of Human Genetics.

[10]  Kou-Juey Wu,et al.  c-Myc Directly Regulates the Transcription of the NBS1 Gene Involved in DNA Double-strand Break Repair * , 2003, Journal of Biological Chemistry.

[11]  B. Vanhaesebroeck,et al.  Activation of phosphoinositide 3-kinase by the NBS1 DNA repair protein through a novel activation motif , 2007, Journal of Molecular Medicine.

[12]  Lucia Y Brown,et al.  Alanine tracts: the expanding story of human illness and trinucleotide repeats. , 2004, Trends in genetics : TIG.

[13]  A. Munnich,et al.  Polyalanine expansions might not result from unequal crossing‐over , 2007, Human Mutation.

[14]  H. Shih,et al.  SUMO modification negatively modulates the transcriptional activity of CREB-binding protein via the recruitment of Daxx. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[15]  P. Robinson,et al.  A molecular pathogenesis for transcription factor associated poly-alanine tract expansions. , 2004, Human molecular genetics.

[16]  A. Munnich,et al.  Molecular consequences of PHOX2B missense, frameshift and alanine expansion mutations leading to autonomic dysfunction. , 2005, Human molecular genetics.

[17]  Kou-Juey Wu,et al.  Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-MYC. , 1999, Science.

[18]  R. Goodman,et al.  CBP/p300 in cell growth, transformation, and development. , 2000, Genes & development.

[19]  A. Munnich,et al.  Germline mutations of the paired-like homeobox 2B (PHOX2B) gene in neuroblastoma. , 2004, American journal of human genetics.

[20]  D. Shannon,et al.  Idiopathic congenital central hypoventilation syndrome: diagnosis and management. American Thoracic Society. , 1999, American journal of respiratory and critical care medicine.

[21]  R. Ravazzolo,et al.  PHOX2B mutations and polyalanine expansions correlate with the severity of the respiratory phenotype and associated symptoms in both congenital and late onset Central Hypoventilation syndrome , 2004, Journal of Medical Genetics.

[22]  S. Mundlos,et al.  The other trinucleotide repeat: polyalanine expansion disorders. , 2005, Current opinion in genetics & development.

[23]  E. Berry-Kravis,et al.  Congenital central hypoventilation syndrome: PHOX2B mutations and phenotype. , 2006, American journal of respiratory and critical care medicine.

[24]  J. Brunet,et al.  Paired‐Like Homeodomain Proteins, Phox2a and Phox2b, Are Responsible for Noradrenergic Cell‐Specific Transcription of the Dopamine β‐Hydroxylase Gene , 1998, Journal of neurochemistry.