Patient mutations alter ATRX targeting to PML nuclear bodies.
ATRX is a SWI/SNF-like chromatin remodeling protein mutated in several X-linked mental retardation syndromes. Gene inactivation studies in mice demonstrate that ATRX is an essential protein and suggest that patient mutations likely retain partial activity. ATRX associates with the nuclear matrix, pericentromeric heterochromatin, and promyelocytic leukemia nuclear bodies (PML-NBs) in a speckled nuclear staining pattern. Here, we used GFP-ATRX fusion proteins to identify the specific domains of ATRX necessary for subnuclear targeting and the effect of patient mutations on this localization. We identified two functional nuclear localization signals (NLSs) and two domains that target ATRX to nuclear speckles. One of the latter domains is responsible for targeting ATRX to PML-NBs. Surprisingly, this domain encompassed motifs IV-VI of the SNF2 domain suggesting that in addition to chromatin remodeling, it may also have a role in subnuclear targeting. More importantly, four different patient mutations within this domain resulted in an approximately 80% reduction in the number of transfected cells with ATRX nuclear speckles and PML colocalization. These results demonstrate that patient mutations have a dramatic effect on subnuclear targeting to PML-NBs. Moreover, these findings support the hypothesis that ATRX patient mutations represent functional hypomorphs and suggest that loss of proper targeting to PML-NBs is an important contributor to the pathogenesis of the ATR-X syndrome.
Bérubé
Nathalie G
NG
Molecular Medicine Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada.
Healy
Jasmine
J
Medina
Chantal F
CF
Wu
Shaobo
S
Hodgson
Todd
T
Jagla
Magdalena
M
Picketts
David J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics*; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D006367:HeLa Cells; D006801:Humans; D045586:Intranuclear Inclusion Bodies / Q000378:metabolism*; D038901:X-Linked Intellectual Disability / Q000235:genetics / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion / Q000235:genetics*; D013577:Syndrome; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism*; D000075924:X-linked Nuclear Protein
D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D004265:DNA Helicases; C092332:ATRX protein, human; D000075924:X-linked Nuclear Protein