A Multiprotein Complex Involved In Atrx Syndrome

ATRX综合征是α -地中海贫血、智力迟钝和多种相关发育异常的组合。ATRX的缺陷基因定位在X染色体上并进行了克隆。同一基因的突变还会导致其他几种形式的x连锁智力迟钝综合症。ATRX基因编码一个含有SWI2/ snf2型dna依赖性atp酶结构域的基因产物。因此，我们假设ATRX可能在atp依赖性染色质重塑复合体中起作用，并参与基因表达的调控。我们与dr。道格·希格斯和理查德·吉本斯，他们克隆了ATRX基因，并发现了其功能的重要特征。通过HeLa提取物的免疫沉淀，我们发现ATRX与转录辅助因子Daxx在一个复合物中。以下证据支持ATRX和Daxx是atp依赖性染色质重塑复合体的组成部分：1)Daxx和ATRX可以通过特异性抗体共同免疫分离；2)通过凝胶过滤分析，一定比例的Daxx与ATRX作为1mda的络合物共分馏；3) ATRX综合征患者细胞提取物中Daxx-ATRX复合物水平相应降低；4) ATRX和Daxx在PML核体中有一定比例的共定位，而Daxx在PML核体中已被发现；5) ATRX复合物显示atp依赖的活性，类似于其他染色质重塑复合物，包括三螺旋DNA位移和单核小体破坏模式的改变。但与先前描述的SWI/SNF或NURD复合物不同，ATRX复合物不会随机化单核小体的DNA相位，这表明它可能以不同的方式重塑染色质。综上所述，结果表明ATRX与Daxx作为一种新的染色质重塑复合体共同起作用。ATR-X综合征的缺陷可能是由该复合体控制的基因表达不当造成的。我们已经确定了几个序列特异性转录因子与Daxx共纯化。我们现在正在研究由这些因子调控的基因是否与ATR-X综合征有关。

ATRX syndrome represents a combination of alpha-thalassemia, mental retardation, and multiple associated developmental abnormalities. The gene defective in ATRX has been localized to the X chromosome and cloned. Mutations in the same gene also cause several other forms of syndromal X-linked mental retardation. The ATRX gene encodes a gene product containing a SWI2/SNF2-type DNA-dependent ATPase domain. Thus, it has been hypothesized that ATRX could function in an ATP-dependent chromatin-remodeling complex and participate in regulation of gene expression. We established a collaboration with Drs. Doug Higgs and Richard Gibbons, who had cloned the ATRX gene, and have discovered important features of its function. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: 1) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; 2) a proportion of Daxx co-fractionates with ATRX as a complex of 1 MDa by gelfiltration analysis; 3) in extract of cells of a patient with ATRX syndrome, the level of the Daxx-ATRX complex is correspondingly reduced; 4) a proportion of ATRX and Daxx colocalize in PML nuclear bodies, where Daxx had previously been found; and 5) ATRX complex displays ATP-dependent activities that resemble those of other chromatin remodeling complexes, including triple helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx as a novel chromatin-remodeling complex. The defects in ATR-X syndrome may result from inappropriate expression of genes controlled by this complex.We have identified several sequence-specific transcription factors that co-purify with Daxx. We are now studying if the genes regulated by these factors could be involved in ATR-X syndrome.