Transcription at the centromere: Opportunity and danger for the maintenance of epigenetic identity

如何从单个细胞中创造出一个有机体是生物学中最令人惊奇的过程之一。从一个受精卵开始，这个细胞将开始分裂多次，产生多细胞动物，植物或真菌。细胞分裂不会停止，一旦完全生长，但继续通过生命的有机体。这对于确保在经历自然细胞死亡或与环境相互作用时保持身体形状和大小至关重要。重要的是，在生物体宏观水平上重要的东西也在细胞水平上发挥作用。细胞的基本任务是发展和保持其特性。细胞的身份是编码在基因组中，它以染色体的形式出现。染色体由染色质长纤维形成，染色质长纤维由DNA组成，DNA与称为组蛋白的蛋白质包装在一起。四种不同的组蛋白H2 A，H2 B，H3和H4形成一个更大的盘状复合物，称为核小体，DNA包裹在核小体周围，使染色质纤维看起来像一串珠子。在细胞周期中，染色体在母细胞中复制，并在细胞分裂时传递给两个子细胞。虽然大多数遗传信息在DNA序列中编码，但人们普遍认为染色质携带的信息是独立于潜在的DNA序列传递的。这种形式的遗传被称为“表观遗传”，可以决定某些基因是否表达或定义染色体特定区域的身份。着丝粒是这些专门化的区域之一。在显微镜下可以看到X形染色体上的收缩，在细胞分裂过程中对染色体的分离是必不可少的。着丝粒的身份是通过组蛋白H3的同源物，即着丝粒特异性组蛋白CENP-A（果蝇中的dCENP-A）的存在来确定的。着丝粒染色质由CENP-A和典型组蛋白H3核小体的散布阵列组成。虽然H3在S期的DNA复制过程中得到补充，但果蝇和人类中CENP-A的加载从有丝分裂晚期到G1以不依赖复制的方式发生。这个过程需要去除所谓的占位符H3-核小体，其在先前的S期期间已经定位在着丝粒DNA序列上。近年来，我们和其他实验室已经提出证据表明，一种称为转录的特定细胞过程可以允许这种染色质重塑发生，并允许CENP-A交换H3。需要直接DNA接触的细胞过程，如DNA复制或转录，诱导大规模染色质重塑事件，以允许DNA和RNA聚合酶的进展。转录在基因组中的主要作用是转录基因并产生编码蛋白质的RNA转录物、转运RNA或核糖体RNA。由于在着丝粒上没有基因，我们假设转录被用来重塑染色质并使核小体不稳定，足以驱逐H3-占位符。这将构成另一个有趣的例子，自然如何通过重新利用现有的工具箱来解决复杂的问题。虽然这是一个有趣的假设，但这一过程仍然没有被理解，并提出了许多问题：着丝粒转录如何区分应该保留的CENP-A核小体和应该删除的占位符H3-核小体？这一过程是如何监管的？在寻找CENP-A的结合伴侣时，我们先前鉴定了Spt 6，一种在转录过程中参与组蛋白再循环的蛋白质。在这里，我们建议调查人类和果蝇Spt 6结合CENP-A和H3组蛋白的分子细节，以及如何在着丝粒转录有助于CENP-A加载。通过研究这一重要的进化保守过程，着丝粒可以作为一个范例来理解表观遗传的潜在机制，以保持细胞的身份。

How to make an organism out of a single cell is one of the most amazing processes in biology. Starting with a single fertilized egg, this cell will start to divide many times to give rise to a multicellular animal, plant or fungus. The cell divisions will not stop once fully grown but continue through the lifetime of the organism. This is essential to ensure that body shape and size are maintained, while experiencing natural cell death or caused by interacting with its environment.Importantly, what is important at the macroscopic level of an organism also plays a role at the cellular level. Developing but also maintaining its identity is an essential task for a cell. Cellular identity is encoded in the genome which comes in the shape of chromosomes. Chromosomes are formed by long fibers of chromatin, made of DNA that is packaged together with proteins called histones. Four different histones H2A, H2B, H3 and H4 form a larger disk-shaped complex called a nucleosome around which the DNA is wrapped giving the chromatin fiber the appearance of beads on a string. During the cell cycle, chromosomes are duplicated in a mother cell and passed on to two daughter cells as cells divide. While most genetic information is encoded in the DNA sequence, it is widely appreciated that chromatin carries information that is passed on independently of the underlying DNA sequence. This form of inheritance is called "epigenetic" and can determine whether certain genes are expressed or define the identity of specific regions of the chromosome. Centromeres are among those specialised regions. They are visible as constrictions in X-shaped chromosomes under the microscope and essential for the separation of chromosomes during cell division.Centromere identity is determined epigenetically through the presence of a homolog of histone H3, the centromere-specific histone CENP-A (dCENP-A in Drosophila). Centromeric chromatin is composed of interspersed arrays of CENP-A and canonical histone H3 nucleosomes. While H3 is replenished during DNA replication in S-phase, loading of CENP-A in Drosophila and humans takes place in a replication-independent manner from late mitosis to G1. This process requires the removal of so-called placeholder H3-nucleosomes, which have been positioned on centromeric DNA-sequences during the previous S-phase. In recent years, we and other labs have presented evidence that one particular cellular process called transcription could allow for this chromatin remodeling to take place and allow the exchange of H3 by CENP-A.Cellular processes that require direct DNA contact like DNA replication or transcription induce large-scale chromatin remodeling events to allow the progression of DNA- and RNA- polymerases. The major role of transcription in the genome is to transcribe genes and produce RNA transcripts encoding proteins, transfer or ribosomal RNA. As there are no genes at the centromere, we hypothesise that transcription is instead used to remodel chromatin and destabilise nucleosomes enough to evict H3-placeholders. This would constitute another fascinating example, how nature solves complex problems by repurposing existing toolkits.Although an intriguing hypothesis, this process is still not understood and raises many questions: How does centromeric transcription distinguish CENP-A nucleosome that should be retained from placeholder H3-nucleosomes that should be removed? How is this process regulated? In a search for binding partners of CENP-A, we previously identified Spt6, a protein involved in recycling histones during transcription. Here, we propose to investigate the molecular details of human and Drosophila Spt6 binding to CENP-A and H3 histones and how transcription at the centromere contributes to CENP-A loading. By studying this essential evolutionary conserved process, centromeres can be used as a paradigm to understand the underlying mechanism of epigenetic inheritance to preserve the identity of the cell.