Sequence features and transcriptional stalling within centromere DNA promote establishment of CENP-A chromatin.
Sequence features and transcriptional stalling within centromere DNA promote establishment of CENP-A chromatin.
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DOI：
10.1371/journal.pgen.1004986
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发表时间：
2015-03
期刊：
影响因子：
4.5
通讯作者：
Allshire RC
中科院分区：
文献类型：
--
作者：
Catania S;Pidoux AL;Allshire RC
着丝粒序列在物种之间并不保守，并且有令人信服的证据表明着丝粒身份的表观遗传调节，其位置由含有组蛋白H3变体CENP-A的染色质的存在决定。巧合的是，在大多数生物体中，CENP-A染色质通常出现在特定序列上。为了研究一级DNA序列对体内CENP-A染色质建立的贡献，我们利用裂殖酵母粟酒裂殖酵母。CENP-ACnp 1染色质通常组装在这些区域性着丝粒内的约10 kb的中央结构域DNA上。我们证明了S.粟酒裂殖酵母CENP-ACnp 1绕过了在建立CENP-ACnp 1染色质中对相邻异染色质的通常要求，并表明中心结构域DNA是从头建立CENP-ACnp 1染色质的优选底物。当多聚化时，2kb的亚区可以建立CENP-ACnp 1染色质并形成功能性着丝粒。随机化2kb序列以产生维持AT含量和预测的核小体定位的序列不能建立CENP-ACnp 1染色质。这些分析表明，来自裂殖酵母着丝粒的中央结构域DNA含有促进CENP-ACnp 1掺入染色质的特定信息。在功能性2kb亚区内的正向和反向链上检测到许多转录起始位点，并鉴定了活性启动子。RNAPII在野生型细胞的中央结构域DNA上富集，但仅检测到低水平的转录物，这与着丝粒DNA转录期间RNAPII停滞一致。当CENP-ACnp 1处于野生型水平时，缺乏参与重新启动转录TFIIS和Ubp 3的因子的细胞在中央结构域DNA上组装CENP-ACnp 1，这表明RNAPII在着丝粒DNA上的持续停滞触发染色质重塑事件，存款CENP-ACnp 1。因此，着丝粒DNA的序列编码特征创造了普遍的低质量RNAPII转录的环境，这是CENP-ACnp 1组装的重要决定因素。这些观察强调了遗传和表观遗传过程在着丝粒建立中的作用。动粒引导染色体的分离，并在染色体的一个特殊区域--着丝粒上组装。DNA被包裹在称为核小体的颗粒周围，核小体含有组蛋白。位于着丝粒的核小体是特化的，并且含有着丝粒特异性组蛋白CENP-A。CENP-A核小体形成了构建动粒的平台。因此，CENP-A和着丝粒功能齐头并进。细胞如何确保CENP-A沉积在着丝粒而不是其他地方还不清楚。我们研究了DNA序列在确定裂殖酵母中着丝粒功能中的作用。我们的观察表明，吸引CENP-A的重要因素不是DNA序列本身，而是序列创造的特定环境。在着丝粒DNA的转录过程中，RNA聚合酶（RNAPII）似乎卡住或停滞。特定的蛋白质-如TFIIS和Ubp 3-已知有助于重新启动RNAPII，使其能够继续转录。我们发现，当细胞缺乏Ubp 3或TFIIS时，CENP-A会沉积在着丝粒序列上。我们认为RNAPII在着丝粒DNA上的持续停滞吸引了有助于存款CENP-A的因子。这项研究强调了DNA序列在为CENP-A组装创造有吸引力的环境中的影响。
Centromere sequences are not conserved between species, and there is compelling evidence for epigenetic regulation of centromere identity, with location being dictated by the presence of chromatin containing the histone H3 variant CENP-A. Paradoxically, in most organisms CENP-A chromatin generally occurs on particular sequences. To investigate the contribution of primary DNA sequence to establishment of CENP-A chromatin in vivo, we utilised the fission yeast Schizosaccharomyces pombe. CENP-ACnp1 chromatin is normally assembled on ∼10 kb of central domain DNA within these regional centromeres. We demonstrate that overproduction of S. pombe CENP-ACnp1 bypasses the usual requirement for adjacent heterochromatin in establishing CENP-ACnp1 chromatin, and show that central domain DNA is a preferred substrate for de novo establishment of CENP-ACnp1 chromatin. When multimerised, a 2 kb sub-region can establish CENP-ACnp1 chromatin and form functional centromeres. Randomization of the 2 kb sequence to generate a sequence that maintains AT content and predicted nucleosome positioning is unable to establish CENP-ACnp1 chromatin. These analyses indicate that central domain DNA from fission yeast centromeres contains specific information that promotes CENP-ACnp1 incorporation into chromatin. Numerous transcriptional start sites were detected on the forward and reverse strands within the functional 2 kb sub-region and active promoters were identified. RNAPII is enriched on central domain DNA in wild-type cells, but only low levels of transcripts are detected, consistent with RNAPII stalling during transcription of centromeric DNA. Cells lacking factors involved in restarting transcription—TFIIS and Ubp3—assemble CENP-ACnp1 on central domain DNA when CENP-ACnp1 is at wild-type levels, suggesting that persistent stalling of RNAPII on centromere DNA triggers chromatin remodelling events that deposit CENP-ACnp1. Thus, sequence-encoded features of centromeric DNA create an environment of pervasive low quality RNAPII transcription that is an important determinant of CENP-ACnp1 assembly. These observations emphasise roles for both genetic and epigenetic processes in centromere establishment. The kinetochore directs the separation of chromosomes and is assembled at a special region of the chromosome—the centromere. DNA is wrapped around particles called nucleosomes, which contain histone proteins. The nucleosomes at centromeres are specialized, and contain the centromere-specific histone CENP-A. CENP-A nucleosomes form the platform upon which the kinetochore is built. Thus, CENP-A and centromere function go hand-in-hand. How the cell ensures that CENP-A is deposited at centromeres and not elsewhere is not well understood. We investigated the role that DNA sequence plays in defining centromere function in fission yeast. Our observations suggest that it is not the DNA sequence per se that is important for attracting CENP-A, but rather, the particular environment that the sequence creates. During transcription of centromeric DNA, RNA polymerase (RNAPII) appears to get stuck or stalled. Particular proteins—such as TFIIS and Ubp3—are known to help restart RNAPII so it can continue transcribing. We found that when cells lack Ubp3 or TFIIS, CENP-A becomes deposited on centromere sequences. We propose that persistent stalling of RNAPII on centromere DNA attracts factors that help deposit CENP-A. This study highlights the influence of DNA sequence in creating an attractive environment for CENP-A assembly.
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影响因子：
4.5
作者：
Chueh, Anderly C.;Northrop, Emma L.;Brettingham-Moore, Kate H.;Choo, K. H. Andy;Wong, Lee H.
通讯作者：
Wong, Lee H.
影响因子：
7.5
作者：
Catania, Sandra;Allshire, Robin C.
通讯作者：
Allshire, Robin C.
影响因子：
3.3
作者：
BAUM, M;NGAN, VK;CLARKE, L
通讯作者：
CLARKE, L
裂殖酵母中着丝粒和端粒的细胞周期依赖性特定定位和聚类。
影响因子：
7.8
作者：
Funabiki, H;Hagan, I;Uzawa, S;Yanagida, M
通讯作者：
Yanagida, M
DOI：
10.1016/j.bbagrm.2011.11.002
发表时间：
2012-03
影响因子：
4.7
作者：
Falk, Samantha J.;Black, Ben E.
通讯作者：
Black, Ben E.