A new fractionation assay, based on the size of formaldehyde-crosslinked, mildly sheared chromatin, delineates the chromatin structure at promoter regions.
A new fractionation assay, based on the size of formaldehyde-crosslinked, mildly sheared chromatin, delineates the chromatin structure at promoter regions.
复制标题
DOI：
10.1093/nar/gkq203
复制
发表时间：
2010-06
影响因子：
14.9
通讯作者：
Schwartz RH
中科院分区：
文献类型：
--
作者：
Ishihara S;Varma R;Schwartz RH
为了探索体内可转录染色质的高级结构，通过染色质与甲醛交联的可及性来评估其局部构型。应用交联和轻度剪切的染色质进行沉降速度离心，然后对 DNA 进行大小分级，使我们能够在生化上区分具有重交联结构和稀疏交联结构的染色质。分离的组分显示出与基因表达谱良好的相关性。启动子区域周围交联较差的基因转录活跃，而启动子区域交联广泛的基因几乎检测不到转录物。对于诱导型基因 Il2，T 细胞受体刺激后启动子的分布发生梯度变化，与激活过程中该位点的结构变化一致。这种转换的动力学先于 DNase I 可及性测定中观察到的染色质变化。因此，这种新的染色质分级分离技术揭示了以前未表征的染色质结构的变化。
To explore the higher order structure of transcribable chromatin in vivo, its local configuration was assessed through the accessibility of the chromatin to crosslinking with formaldehyde. The application of crosslinked and mildly sheared chromatin to sedimentation velocity centrifugation followed by size-fractionation of the DNA enabled us to biochemically distinguish between chromatin with heavily versus sparsely crosslinkable structures. The separated fractions showed a good correlation with gene expression profiles. Genes with poor crosslinking around the promoter region were actively transcribed, while transcripts were hardly detected from genes with extensive crosslinking in their promoter regions. For the inducible gene, Il2, the distribution of the promoter shifted in the gradient following T-cell receptor stimulation, consistent with a change in structure at this locus during activation. The kinetics of this switch preceded the chromatin change observed in a DNase I accessibility assay. Thus, this new chromatin fractionation technique has revealed a change in chromatin structure that has not been previously characterized.
登录
查看更多内容
DOI：
10.1006/meth.1999.0879
发表时间：
1999-11-01
期刊：
METHODS-A COMPANION TO METHODS IN ENZYMOLOGY
影响因子：
--
作者：
Kuo, MH;Allis, CD
通讯作者：
Allis, CD
影响因子：
5.3
作者：
Chen, XX;Wang, J;Shannon, MF
通讯作者：
Shannon, MF
影响因子：
13.8
作者：
Orlando, V
通讯作者：
Orlando, V
影响因子：
5.6
作者：
CAPLAN, A;KIMURA, T;ALLAN, J
通讯作者：
ALLAN, J
影响因子：
5.3
作者：
Schwartz, YB;Kahn, TG;Pirrotta, V
通讯作者：
Pirrotta, V