性染色体在哺乳动物X失活

两个X染色体在哺乳动物雌性功能非常不同。X染色体包含超过1000基因,而Y染色体基因相对贫穷。胎盘类哺乳动物这提出了一个挑战,他们需要(大约)平衡表达式X染色体的男性(XY)和女性(XX)细胞。因此,哺乳动物的解决方案灭活一个X染色体女性在早期发展通过装配的一种独特形式染色质这很大程度上沉默基因表达式。以来的第一个图片不活跃的X染色体发表在1949年,科学家们研究了失活过程详细,但是他们发现X染色体具有独特的监管区域,称为X-inactivation中心,对失活至关重要。

图1

不难选出的不活跃的X染色体间期细胞。与其他染色体,不活跃的X从未完全decondenses在间期。因此,不活跃的X出现人口彩色结构在间期核女性的细胞。穆雷巴尔和艾瓦特伯特伦提供的第一批图像不活跃的X在1949年的一项研究中,他们指出以下几点:

“性的体细胞一样高度分化的神经元可能发现没有更复杂的设备比复合显微镜后染色组织的常规尼氏小方法……核结构的不同神经元的成年男性和女性之间猫取决于程度的第二个身体的发展,这是远小于核仁。”

巴尔与伯特伦称之为第二身体,我们现在称之为巴氏小体核仁的卫星,因为它是经常位于旁边的核仁。他们的数据表明,巴氏小体是成熟的女性(图1),但不发达男性(图2)。因此,两人猜测“核仁的卫星可能来源于异染色质的性染色体有先见之明。”研究人员现在欣赏这些观察,即使巴尔和伯特伦无法认识到这颗卫星是一个X染色体。

十年后,Susumu Ohno和他的同事们(Ohnoet al。,1959)提供了更多的证据表明,巴氏小体是一个X染色体。通过系统地比较男性和女性肝细胞在不同的点细胞周期,他们发现女性细胞包含一个高度浓缩的染色体缺失在染色体男性细胞的扩散。此外,他们指出,三倍体细胞这种凝聚结构的额外副本。从数据,Ohno等。推断巴氏小体,或性染色质,可能是一个浓缩的X染色体。最终证明其身份会之后,科学家研制的分子探针,可以用来识别特定的染色体原位杂交。

图2

在1960年代早期,研究员玛丽·里昂建立在大野耐一的细胞学观察当她制定X-inactivation假说(1961年里昂,1962)。里昂是一个鼠标遗传学家激起了他的女性,而不是男性,老鼠斑点外套颜色时杂合的为位于性染色体的基因在外套的颜色。当时,几个x连锁基因与外套颜色,和调查人员能够执行必要的繁殖实验了解外套颜色的继承。结合遗传和细胞学的数据,里昂解释“性染色质”是一个不活跃的X染色体基因的表现。她可以解释雌性老鼠的斑驳的外观如下:

“这是表明这马赛克表型是由于一个或另一个X染色体的失活在胚胎发育的早期。如果这是真的,色素细胞细胞的染色体携带的后裔突变体基因灭活会引起的normal-coloured补丁和染色体携带正常基因灭活会引起mutant-coloured补丁”(里昂,1961)。

里昂的假设的一个重要元素是失活过程的随机性质,从而影响母体遗传来的或从父本继承了X染色体。失活也解释了为什么XO雌性老鼠能够生存一个X染色体。在这个问题上,在她的第二篇论文(1962),里昂将她的分析扩展到其他物种,包括猫和人类,结束X失活一般适用于哺乳动物。

图3

细节图

里昂提出X失活,之后的几十年里,研究人员产生了大量的数据与理论一致,但控制失活的因素仍然难以捉摸。老鼠和人类细胞的细胞遗传学研究与删除或重新安排X染色体指出存在的一个独特的X染色体区域,称为X-inactivation中心(缩写XIC或XIC在人类和小鼠,分别)X-inactivation过程发生的关键(Rastan和罗伯逊,1985)。通过比较一系列缺失染色体,调查人员能够缩小XIC的位置问的X染色体的一部分。最终的一个关键因素映射的XIC rodent-human的可用性混合动力细胞系,改变人类的X染色体。这些线已经被融合派生人类细胞,获得患者进行不同的X染色体易位或删除啮齿动物细胞组织培养。混合细胞系,这个过程通常只包含几个人类染色体,可以确定使用chromosome-specific标记。

使用这种方法,布朗和他的同事们(棕色et al。,1991)研究了杂种细胞系中含有部分人类X染色体的片段,问是否有特定区域的X染色体失活所必需的。灭活的X染色体可以区别于其他染色体在细胞相对较晚复制在S期,这是一个异染色质的特征。图3显示了染色体组型从四个细胞系的重新排列人类X染色体,而正常的左边的X染色体。从这个图中,从棕色et al。的文章,很明显,改变X染色体有广泛的重组,以及它们之间,几乎没有共同点。尽管如此,所有的改变X染色体仍失活。通过仔细观察发生在原始的断点易位或删除事件,布朗和他的同事们能够识别共同的地区的X染色体的长臂上出现在所有染色体进行了失活。然后他们使用原位杂交和一系列的X染色体DNA调查进一步定义共同的边界地区,这与Xq13的远端部分区域。的同源地区,Xic,随后被确认在鼠标,科学家使用转基因小鼠模型建立的要求这一地区在X失活。

XIST gene in males, females and somatic cell hybrids.", "true", "Figure 4", "Slot blot of total cellular RNA isolated from human lymphoblastoid cell lines or mouse-human somatic cell hybrids retaining either the active or inactive human X chromosome, hybridized with the 14A XIST cDNA proble. The probe hyrbidizes only to RNA samples from cell lines which contain an inactive X chromosome.", "true", "All rights reserved.", '662', '557', '//www.scienovate.com');">

XIST gene in males, females and somatic cell hybrids.", "true", "Figure 4", "Slot blot of total cellular RNA isolated from human lymphoblastoid cell lines or mouse-human somatic cell hybrids retaining either the active or inactive human X chromosome, hybridized with the 14A XIST cDNA proble. The probe hyrbidizes only to RNA samples from cell lines which contain an inactive X chromosome.", "true", "All rights reserved.", '662', '557', '//www.scienovate.com');">图4

很意外地是,布朗等。从XIC地区还发现了一个不同寻常的记录。特别是,而努力克隆另一个X染色体上的基因,他们发现XIC地区特异表达的基因在染色体进行了失活(棕色等,1991 a, b)。根据这本小说性质,他们给这个基因XIST“X不活跃的特定的记录。”To demonstrate that theXIST基因表达特别的不活跃的X,研究人员做了一个定量的比较XIST转录在正常和非整倍体人类细胞系,以及rodent-human细胞系。图4显示了实验的数据,使用DNA杂交来测量的相对数量XIST核糖核酸在总RNA的样品准备的各种细胞系。在这个实验中,一个乐队的强度约是相称的XISTRNA在样本。RNA样本的来源是显示在右边。这些数据,清楚的说明XIST表达在正常(46,XX)女性,但不是在正常男性(46,XY)。在非整倍体细胞系与47 49染色体,的数量XIST记录额外X染色体的数目成正比。rodent-human混合细胞系被分成Xa和习近平类,反映人类X是否已经被证明是积极或不活跃,分别。的结果,很明显,XISTRNA产生只有在不活跃的和不活跃的X在rodent-human混合细胞。一个控制实验表明Xist小鼠染色体的成绩单就不会产生混合线。(人类XIST对应于鼠标Xist)。综上所述,结果,清楚的说明XIST是转录从灭活X染色体。

Xist transcription in embryonic stem cells.", "true", "Figure 5", "Patterns of Xist RNA expression in female ES cells undergoing differentiation using RNA fluorescence in situ hybridization. The left panel shows that undifferentiated ES cells have two punctate Xist RNA signals, representing the presence of unstable Xist transcripts at the site of transcription on both (active) X chromosomes. The middle panel shows that, upon differentiation, Xist RNA from one of the two alleles becomes stabilized and coats the X chromosome that is to be inactivated in cis. The X chromosome that remains active continues to express Xist in its unstable form. The right panel shows that, in fully differentiated cells, Xist RNA coats the inactive X chromosome and the Xist gene on the active X has been silenced.", "true", "All rights reserved.", '600', '209', '//www.scienovate.com');">

Xist transcription in embryonic stem cells.", "true", "Figure 5", "Patterns of Xist RNA expression in female ES cells undergoing differentiation using RNA fluorescence in situ hybridization. The left panel shows that undifferentiated ES cells have two punctate Xist RNA signals, representing the presence of unstable Xist transcripts at the site of transcription on both (active) X chromosomes. The middle panel shows that, upon differentiation, Xist RNA from one of the two alleles becomes stabilized and coats the X chromosome that is to be inactivated in cis. The X chromosome that remains active continues to express Xist in its unstable form. The right panel shows that, in fully differentiated cells, Xist RNA coats the inactive X chromosome and the Xist gene on the active X has been silenced.", "true", "All rights reserved.", '600', '209', '//www.scienovate.com');">图5:Xist在胚胎干细胞转录。

Xist transcription in embryonic stem cells.", "true", "Figure 5", "Patterns of Xist RNA expression in female ES cells undergoing differentiation using RNA fluorescence in situ hybridization. The left panel shows that undifferentiated ES cells have two punctate Xist RNA signals, representing the presence of unstable Xist transcripts at the site of transcription on both (active) X chromosomes. The middle panel shows that, upon differentiation, Xist RNA from one of the two alleles becomes stabilized and coats the X chromosome that is to be inactivated in cis. The X chromosome that remains active continues to express Xist in its unstable form. The right panel shows that, in fully differentiated cells, Xist RNA coats the inactive X chromosome and the Xist gene on the active X has been silenced.", "true", "All rights reserved.", '600', '209', '//www.scienovate.com');">的模式XistRNA表达女性ES细胞进行分化使用RNA荧光原位杂交。左边的面板显示,未分化的胚胎干细胞有两个点状的XistRNA信号,代表不稳定的存在Xist转录的转录(主动)X染色体。中间面板显示,在分化,Xist两个等位基因的RNA从一个变得稳定和外套的X染色体在独联体被灭活。仍然活跃的X染色体继续表达Xist其不稳定的形式。右边的面板显示了,在完全分化的细胞,XistRNA外套不活跃的X染色体和Xist基因活性X一直沉默。

©2001自然出版集团会员,p &听到,e . x染色体失活:计数、选择和启动。自然遗传学评论 2,62 (2001)。保留所有权利。

从最初的发现XIST科学家怀疑,XIST成绩单有不同寻常的特性。布朗et al。已经观察到多个XIST成绩单是由选择拼接在细胞。然而,有许多停止密码子在所有潜在的阅读框架,使它不可能XIST成绩单翻译在细胞。事实上,本地化的XIST记录细胞的荧光原位杂交(鱼证实这些怀疑,指着一个独特的功能XIST记录。克莱姆森和他的同事(1996)指出,与其他单份基因的转录,XIST记录形成的散射集群,仍在正常女性人类成纤维细胞的细胞核。的数量XIST集群的数量似乎是一样的不活跃的X染色体,暗示XIST记录保持身体与不活跃的X染色体。这个假设被证实时,调查人员同时杂化XIST探针与探针X染色体的特定的集合。XIST集群总是与X染色体探针硝唑,这也给了集群的外观。此外,在女性细胞非整倍体的数量XIST集群总是一个小于X染色体集群的数量,符合一个活跃的X染色体的存在。因此,研究人员得出结论XIST记录“画”不活跃的X染色体长度。他们还指出,XIST没有直接交互与X染色体DNA,因为它是不被酶退化的DNA。基于这些发现,他们建议XIST成绩单发挥建筑作用定义不活跃的X染色体的核领域,这与当前的知识独特的染色质活性X。

定义的角色XISTRNA在X失活,研究人员需要一个实验模型这可能是转基因。因此,实验工作与X失活与实验室老鼠已经几乎完全执行。小鼠胚胎干细胞(ES)细胞提供了宝贵的模型为研究初始事件X失活(马丁et al。,1978年),发生在早期的哺乳动物胚胎。ES细胞是来源于早期胚胎内细胞团,这些细胞保持多能,直到它们诱导分化,之后,X染色体进行随机失活。的存在Xist成绩单在ES细胞可以通过荧光可视化原位杂交(鱼)。图5文件的变化Xist成绩单伴随ES细胞分化。左边的面板显示了一个未分化的胚胎干细胞中Xist转录上能被探测到孕产妇和父亲的X染色体。在分化的中间阶段(中间面板),转录从两个X染色体仍在发生,但是Xist有选择地记录外套一条X染色体。然后,当胚胎干细胞完全分化(右面板),Xist成绩单的外套不活跃的X和Xist转录不再检测到从其他染色体。因此,的时机Xist转录与基本的作用是一致的独联体失活,但其他问题出现。为什么,例如,是谁Xist成绩单不再检测到活跃的X细胞分化后?如何确定哪些X染色体细胞灭活?事实证明,Xist只有一个活动由Xic编码。我们现在知道Xic是复杂的轨迹包括其他序列参与计算X染色体和决定哪些染色体灭活。

cis-inactivation during the initiation of X inactivation. Genes are shown in bold, with the direction of transcription indicated by the arrow. Brx (brain X-linked), Tsx (testis X-linked) and Cdx4 (Caudal-4) are genes that lie within the Xic that do not have, at least for the moment, any defined X-inactivation function. The 2.1(2)P region shows differential histone H4 hyperacetylation in undifferentiated female and male embryonic stem (ES) cells and has been suggested as a possible regulatory element in X inactivation. P1 and P2 are the somatic Xist promoters, and P0 is a postulated Xist promoter in undifferentiated ES cells and early embryos. S12 and S19 are ribosomal protein pseudogenes found 5' to Xist in the mouse Xic. The deletions used to dissect the function of different elements in the Xic are shown as black lines. Blue bars indicate the regions that have been implicated in specific functions. Effects on choice and counting have not so far been distinguished in the regions indicated by light bars. The terminal two Xist exons, lying within the 65-kb deletion, have no effect on counting or choice.", "true", "All rights reserved.", '600', '361', '//www.scienovate.com');">

cis-inactivation during the initiation of X inactivation. Genes are shown in bold, with the direction of transcription indicated by the arrow. Brx (brain X-linked), Tsx (testis X-linked) and Cdx4 (Caudal-4) are genes that lie within the Xic that do not have, at least for the moment, any defined X-inactivation function. The 2.1(2)P region shows differential histone H4 hyperacetylation in undifferentiated female and male embryonic stem (ES) cells and has been suggested as a possible regulatory element in X inactivation. P1 and P2 are the somatic Xist promoters, and P0 is a postulated Xist promoter in undifferentiated ES cells and early embryos. S12 and S19 are ribosomal protein pseudogenes found 5' to Xist in the mouse Xic. The deletions used to dissect the function of different elements in the Xic are shown as black lines. Blue bars indicate the regions that have been implicated in specific functions. Effects on choice and counting have not so far been distinguished in the regions indicated by light bars. The terminal two Xist exons, lying within the 65-kb deletion, have no effect on counting or choice.", "true", "All rights reserved.", '600', '361', '//www.scienovate.com');">图6:X-inactivation中心。

cis-inactivation during the initiation of X inactivation. Genes are shown in bold, with the direction of transcription indicated by the arrow. Brx (brain X-linked), Tsx (testis X-linked) and Cdx4 (Caudal-4) are genes that lie within the Xic that do not have, at least for the moment, any defined X-inactivation function. The 2.1(2)P region shows differential histone H4 hyperacetylation in undifferentiated female and male embryonic stem (ES) cells and has been suggested as a possible regulatory element in X inactivation. P1 and P2 are the somatic Xist promoters, and P0 is a postulated Xist promoter in undifferentiated ES cells and early embryos. S12 and S19 are ribosomal protein pseudogenes found 5' to Xist in the mouse Xic. The deletions used to dissect the function of different elements in the Xic are shown as black lines. Blue bars indicate the regions that have been implicated in specific functions. Effects on choice and counting have not so far been distinguished in the regions indicated by light bars. The terminal two Xist exons, lying within the 65-kb deletion, have no effect on counting or choice.", "true", "All rights reserved.", '600', '361', '//www.scienovate.com');">总结已知的元素和地区X-inactivation中心(Xic)认为影响选择,计数独联体在X失活的起始失活。基因以粗体显示,转录箭头所示的方向。Brx(大脑x连锁),Tsx(睾丸x连锁)和Cdx4(Caudal-4)基因位于Xic没有,至少目前,任何X-inactivation函数定义。2.1 (2)P地区显示差组蛋白H4 hyperacetylation男女在未分化的胚胎干细胞(ES)细胞和被建议作为一个可能的监管元素X失活。P1和P2是体细胞Xist发起人,P0是一个假设Xist启动子在未分化的胚胎干细胞和早期胚胎。S12和S19核糖体蛋白伪基因发现5 'Xist在鼠标Xic。用于解剖的删除功能的不同元素Xic显示为黑色线条。蓝条指示的区域与特定的功能。影响选择和计算,迄今没有被光酒吧表示杰出的区域。终端两个Xist外显子,躺在65 kb的删除,没有影响计算或选择。

©2001自然出版集团会员,p &听到,e . x染色体失活:计数、选择和启动。自然遗传学评论 2,61年。保留所有权利。

cis-inactivation during the initiation of X inactivation. Genes are shown in bold, with the direction of transcription indicated by the arrow. Brx (brain X-linked), Tsx (testis X-linked) and Cdx4 (Caudal-4) are genes that lie within the Xic that do not have, at least for the moment, any defined X-inactivation function. The 2.1(2)P region shows differential histone H4 hyperacetylation in undifferentiated female and male embryonic stem (ES) cells and has been suggested as a possible regulatory element in X inactivation. P1 and P2 are the somatic Xist promoters, and P0 is a postulated Xist promoter in undifferentiated ES cells and early embryos. S12 and S19 are ribosomal protein pseudogenes found 5' to Xist in the mouse Xic. The deletions used to dissect the function of different elements in the Xic are shown as black lines. Blue bars indicate the regions that have been implicated in specific functions. Effects on choice and counting have not so far been distinguished in the regions indicated by light bars. The terminal two Xist exons, lying within the 65-kb deletion, have no effect on counting or choice.", '600','//www.scienovate.com', "The X inactivation center (Xic) is displayed as a long yellow bar with several genes and regulatory elements placed along this portion of the chromosome. Starting on the left, a small region that may be a regulatory element in X inactivation is designated 2.1(2)P and is represented by a short black line above the Xic. Next, the promoter P0 is represented by a right-facing arrow above the Xic. This is followed by two pseudogenes, S12 and S19, represented by vertical black lines within the Xic. Another promoter, P1/P2, is represented by a right-facing arrow above the Xic. P1/P2 is immediately followed by the Xist transcript, which is represented within the Xic as a red bar. Farther down the chromosome, the gene DXPas34 is represented within the Xic as a short grey bar. It is followed immediately by the TsiX promoter, which is represented by a left-facing arrow below the Xic. On the right half of the Xic, three genes are labeled: Tsx, Brx, and Cdx4. Each gene is represented within the Xic as a grey bar with a left-facing arrow.Below the Xic diagram are black lines labeled 1 through 5 that represent portions of the chromosome that were deleted to test the functions of different elements of the Xic. Bar 1 corresponds to P1/P2 and the 5-prime portion of Xist. Bar 2 corresponds to an internal portion of the Xist gene. Bar 3 corresponds to DXPas34 and the TsiX promoter. Bar 4 corresponds to the 3-prime portion of DXPas34, the TsiX promoter, and a small, unlabeled portion of the Xic. Bar 5 extends from the 3-prime portion of the Xist gene to the beginning of the Brx gene.Blue bars under the Xic diagram indicate that Xist, DXPas34, and the 3-prime portion of Xic, including Cdx4, play a role in choice; the middle portion of the Xic, including part of DXPas34, Tsx, and several unlabeled portions, play a role in counting; and the Xist gene plays a role in Cis-inactivation.")" class="inlineLinks">细节图

的时机Xist转录和涂层的不活跃的X染色体是一致的,但是不能证明,要求Xist在X失活。以确定是否Xist确实是需要X失活,研究人员开发了小鼠模型,正常吗Xist基因灭活。一分钱等。(1996)构建女性的ES细胞系Xist在一个X染色体上的基因被删除。这些特殊的胚胎干细胞多态多个x连锁基因,因此调查人员能够识别特定的染色体,灭活细胞分化。通过使用allele-specific探针和x染色体相关记录,研究人员发现,染色体的功能Xist基因总是灭活细胞分化。染色体的变异Xist不再受失活的基因。因此,X染色体的失活需要的功能Xist基因在同一染色体。

的生理后果与损失有关Xist函数被Marahrens进一步探讨et al。(1997)生成的基因敲除老鼠缺乏Xist活动。有趣的男性和女性之间的差异观察小鼠在这些实验中,符合X失活在早期发展的重要性。不足为奇的是,雄性小鼠表型正常,因为X失活不发生在大多数男性组织。唯一的例外是男性生殖细胞系,所以研究人员注意到,男性肥沃和产生精子的能力施肥鸡蛋。两种截然不同的结果观察雌性老鼠,取决于非功能Xist基因是遗传自母亲或父亲。女性继承了缺陷Xist从他们的母亲的表型正常,maternal-specific等位基因在所有组织中都有表达,表明父亲的X被灭活。女性继承了缺陷Xist从他们的父亲,然而,发育迟缓和死亡的胚胎。表达分析表明,产妇X在所有的胚胎细胞灭活,但是,无论是X染色体灭活滋养层,导致胎盘的胚外组织。归因于缺乏杀伤力剂量补偿滋养层,是父亲的X通常是灭活的地方。

累积的结果从这些和其他许多实验证实Xist不仅是必要的,但也足够了吗独联体X染色体的失活。然而,我们现在知道,Xist不能归因于Xic控制所有的功能。Xic内的其他序列计算X染色体的数量很重要,选择X灭活。图6总结元素和地区Xic被牵连的选择,计算独联体失活函数。(五个删除的位置被用来映射这些函数是黑色线条1 - 5所示)。请注意,Xist轨迹中只占一小部分的更大的Xic,已功能映射到450对碱基的X染色体。特别感兴趣的一个特性是40-kilobase反义转录起始位点Tsix,这是表示从active X和已被证明是必不可少的染色体的选择。今天,研究人员继续研究这个复杂的Xic地区,目前仍不完全清楚。

巴尔与伯特伦六十年后第一次描述了女性的细胞核异色的身体,我们有了解背后的分子机制X失活。这些事件的模型如图7所示。绑定的XISTRNA,是本文的重点,被认为是一个早期事件在X染色体灭活。XISTRNA合成的利差从其网站XIC沿着不活跃的x失活涉及macroH2A的招聘更稳定组蛋白和各种变体沉默复合物,以及活性染色质组蛋白修饰常见(会员&听到,2001)。终点是一个不活跃的X染色质结构,是独一无二的。

Xist RNA is expressed in an unstable form (dotted red lines) and the postulated blocking factor(s) (red) prevents Xist upregulation and/or its association with the chromosome in cis. b) Xist RNA becomes upregulated through stabilization, transcriptional upregulation or release of the blocking factor. LINEs might participate in the spreading process in some way either through association with nucleoprotein complexes including Xist or by a mechanism such as Repeat-Induced Gene Silencing (RIGS). c) Stabilized Xist RNA coats the X chromosome before its inactivation. d) Transcriptional silencing of genes on the X chromosome occurs as a result of Xist RNA coating using an unknown mechanism and is rapidly followed by a shift to asynchronous replication timing of the X chromosome. e) Chromatin modifications, such as the histone deacetylation and methylation of promoters of X-linked genes, as well as the recruitment of the histone variant macroH2A, presumably transform the Xist RNA-coated chromosome into a stably inactive and condensed chromatin state.", "true", "Used with permission. All rights reserved.", '700', '1136', '//www.scienovate.com');">

Xist RNA is expressed in an unstable form (dotted red lines) and the postulated blocking factor(s) (red) prevents Xist upregulation and/or its association with the chromosome in cis. b) Xist RNA becomes upregulated through stabilization, transcriptional upregulation or release of the blocking factor. LINEs might participate in the spreading process in some way either through association with nucleoprotein complexes including Xist or by a mechanism such as Repeat-Induced Gene Silencing (RIGS). c) Stabilized Xist RNA coats the X chromosome before its inactivation. d) Transcriptional silencing of genes on the X chromosome occurs as a result of Xist RNA coating using an unknown mechanism and is rapidly followed by a shift to asynchronous replication timing of the X chromosome. e) Chromatin modifications, such as the histone deacetylation and methylation of promoters of X-linked genes, as well as the recruitment of the histone variant macroH2A, presumably transform the Xist RNA-coated chromosome into a stably inactive and condensed chromatin state.", "true", "Used with permission. All rights reserved.", '700', '1136', '//www.scienovate.com');">图7