Genome-wide roles of Foxa2 in directing liver specification.-猫眼课题宝

DOI：

10.1093/jmcb/mjs037

发表时间：

2012-12

期刊：

Journal of molecular cell biology

影响因子：

5.5

通讯作者：

中科院分区：

生物学1区

文献类型：

作者：

Chenhuan Xu;Xiaowen Lu;Eric Chen;Zhiying He;Borjigin Uyunbilig;Guangpeng Li;Yue Ma;Lijian Hui;Bin Xie;Yuan Gao;Xiaoyan Ding;Yi-Ping Hu;Ping Hu;Jingxian Han;Xin Wang

尊敬的编辑，Foxa转录因子家族成员，即Foxa 1、Foxa 2和Foxa 3，在指导肝脏分化和维持肝脏稳态中起着至关重要的作用（Le Lay和Kaestner，2010; Zaret和卡罗尔，2011）。它们早在胚胎定形内胚层（DE）阶段就在几个关键时间点起作用以调节肝分化（Gualdi等人，1996年）。Foxa 1和Foxa 2两者的组合对于前肠DE细胞的形成和胚胎肝发育期间的进一步肝芽分化是必需的（Lee等人，2005年）。Foxa蛋白可以结合Alb 1增强子的浓缩染色质，并在转录起始前诱导局部染色质松弛（Cirillo et al.，2002年）。转录激活前Foxa与Alb 1增强子的结合通过标记染色质决定发育能力。因此，提出Foxa蛋白作为DE中的先锋因子，在新生肝脏中实际转录激活之前启动肝脏基因（Zaret和卡罗尔，2011）。尽管使用ChIP测序（ChIP-seq）技术在成体肝细胞中广泛绘制了Foxa 2结合谱（Wederell等人，2008; Tuteja等人，2009; Wallerman等人，2009;霍夫曼等人，2010），尚未获得Foxa蛋白在胚胎DE阶段的结合谱。已知Foxa家族的功能在胚胎肝发育阶段和发育后阶段之间存在显著差异（Cirillo et al.，2002; Li等人，2011年）。因此，对FOXA在DE阶段结合的系统分析将有助于阐明其在肝发生早期的功能。我们首先建立了一个方案，以有效地分化ES细胞DE细胞，然后早期肝细胞。通过qPCR和FACS试验以及体外肝分化分析证实DE细胞命运定型（补充图S1）。使用获得的DE细胞通过ChIP-seq对Foxa 2结合位点进行定位。我们获得了420万个读段，并且在将这些读段与小鼠基因组比对并仅检索独特读段之后，通过基于模型的ChIP-seq分析（MACS）程序产生了36675个峰（图1A）（Zhang et al. 2008年）。为了验证峰，进行了ChIP-qPCR测定。30个随机选择的峰位点中有28个为Foxa 2结合阳性，而所有21个随机选择的非峰位点均为阴性（补充图S2 A和B），证明了ChIP-seq结果的特异性。基序分析显示所有五个最高得分的基序与先前记录的Foxa 2结合位点一致（补充图S2 C）（Tuteja等人，2009;霍夫曼等人，2010年）。在将富集的峰定位到最近的RefSeq基因的+50kb转录起始位点后，在DE中鉴定8495个基因为Foxa 2的候选靶基因。为了获得关于Foxa 2如何影响其靶基因表达的全基因组视图，从分化过程中的三个时间点采集样品用于微阵列分析：第0天代表ES细胞（无Foxa 2表达），第5天代表DE细胞，第7天代表早期肝细胞。用微阵列数据集进行两个比较：（i）ES细胞和DE细胞之间;和（ii）DE细胞和早期肝细胞之间（图1B）。此外，将从上述两个比较中获得的上调基因定位到通过ChIP-seq鉴定的Foxa 2靶基因库中（图1B）。794个Foxa 2结合的基因在DE阶段后的肝细胞阶段显示出更高的转录水平，尽管Foxa 2的所有DNA结合都发生在DE阶段。如基因表达的参数分析（PAGE）所示，这些基因的晚期激活具有统计学显著性（补充表S1）（Kim和Volsky，2005）。我们的结果表明，许多Foxa 2靶基因的激活确实发生在Foxa 2结合之后，并揭示了在肝分化过程中启动子/增强子占据和立即转录激活之间广泛存在的解偶联，这与先前对Alb 1基因激活的观察一致（Gualdi等人，1996年）的报告。以前，认为Foxa结合与人癌细胞系中的组蛋白H3赖氨酸4二甲基化（H3 K4 me 2）相关（Lupien等人，2008年）。为了研究在DE阶段Foxa 2的结合是否也与某些组蛋白修饰有关，我们评估了DE细胞中Foxa 2靶基因的组蛋白修饰状态。在DE阶段启动子/增强子被Foxa 2占据，并且在早期肝细胞阶段发生转录激活的基因，如Afp、Ttr和其他9个基因，被归类为“晚期基因”。（补充图S3 A和C）;在DE阶段表达水平伴随Foxa 2结合上调的基因，如Foxa 2、Gata 4和9个其它基因，被归类为“早期基因”（补充图S3 B和D）。首先检查DE细胞中所有这些基因的组蛋白H3乙酰化（H3 ac）状态。Foxa 2结合位点周围的H3 ac只能在“早期基因”上检测到，但在“晚期基因”上不能检测到（补充图S4 A）。接下来，检查这些基因上的组蛋白H3甲基化状态。检测到高水平的H3 K4 me 2和H3 K27 me 3 420| Journal of Molecular Cell Biology（2012），4，420-422 doi：10.1093/jmcb/mjs 037在线发表2012年6月28日

Dear Editor, Members of Foxa transcription factor family, namely Foxa1, Foxa2, and Foxa3, play crucial roles in guiding hepatic differentiation and hepatic homeostatic maintenance (Le Lay and Kaestner, 2010; Zaret and Carroll, 2011). They act at several critical time points to regulate hepatic differentiation as early as embryonic definitive endoderm (DE) stage (Gualdi et al., 1996). Combination of both Foxa1 and Foxa2 was required for the formation of foregut DE cells and the further liver bud differentiation during embryonic hepatic development (Lee et al., 2005). Foxa proteins can bind the condensed chromatin of Alb1 enhancer and induce local chromatin relaxation prior to the initiation of transcription (Cirillo et al., 2002). The binding of Foxa on Alb1 enhancer before transcription activation determined the developmental competence by premarking the chromatin. Thus, Foxa proteins were proposed to behave as pioneer factors in DE to prime hepatic genes prior to their actual transcription activation in nascent liver (Zaret and Carroll, 2011). Although Foxa2 binding profiles were extensively mapped in adult liver cells using ChIP-sequencing (ChIP-seq) technology (Wederell et al., 2008; Tuteja et al., 2009; Wallerman et al., 2009; Hoffman et al., 2010), binding profile of Foxa proteins at the embryonic DE stage has not been obtained yet. Functions of Foxa family have been known with significant differences between embryonic hepatic developing stage and post-developing stage (Cirillo et al., 2002; Li et al., 2011). Therefore, a systematic analysis of Foxa binding at the DE stage will help elucidate its functions at early hepatogenesis stage. We first established a protocol to efficiently differentiate ES cells to DE cells and then early hepatic cells. DE cell fate commitment was confirmed by qPCR and FACS assays and in vitro hepatic differentiation analysis (Supplementary Figure S1). The obtained DE cells were used to map Foxa2 binding sites by ChIP-seq. We obtained 4.2 million reads, and 36675 peaks were generated by Model-based Analysis of ChIP-seq (MACS) program after aligning these reads to the mouse genome and retrieving only the unique reads (Figure 1A) (Zhang et al., 2008). To verify the peaks, ChIP-qPCR assays were performed. Twenty-eight out of 30 randomly selected peak sites were positive for Foxa2 binding, while all 21 randomly selected non-peak sites were negative (Supplementary Figure S2A and B), proving the specificity of the ChIP-seq results. Motif analysis showed that all of the five top scored motifs were consistent with previously documented Foxa2 binding sites (Supplementary Figure S2C) (Tuteja et al., 2009; Hoffman et al., 2010). After mapping the enriched peaks to + 50 kb of transcription start sites of the nearest RefSeq genes, 8495 genes were identified as the candidate target genes for Foxa2 in DE. In order to gain a genome-wide view on how Foxa2 affected the expression of its target genes, samples from three time points during differentiation process were taken for microarray analysis: day 0 representing ES cells (no Foxa2 expression), day 5 representing DE cells, and day 7 representing early hepatic cells. Two comparisons were made with the microarray data sets: (i) between ES cells and DE cells; and (ii) between DE cells and early hepatic cells (Figure 1B). Furthermore, the up-regulated genes obtained from the above two comparisons were mapped to the pool of Foxa2 target genes identified by ChIP-seq (Figure 1B). Seven hundred and ninety-four Foxa2-bound genes displayed higher transcription levels at the hepatic cell stage after the DE stage, though all DNA binding by Foxa2 happened at the DE stage. Late activation of these genes was statistically significant as shown by parametric analysis of gene expression (PAGE) (Supplementary Table S1) (Kim and Volsky, 2005). Our results indicated that activation of many Foxa2 target genes indeed happened later than the Foxa2 binding and revealed a widely existing uncoupling between promoter/ enhancer occupation and immediate transcription activation during the process of hepatic differentiation, consistent with previous observations made on Alb1 gene activation (Gualdi et al., 1996). Previously, Foxa binding was suggested to correlate with histone H3 lysine 4 dimethylation (H3K4me2) in human cancer cell lines (Lupien et al., 2008). In order to investigate whether the binding of Foxa2 at the DE stage could also be related to certain histone modifications, we assessed the histone modification status on Foxa2 target genes in DE cells. Genes such as Afp, Ttr, and nine other genes, whose promoter/enhancers were occupied by Foxa2 at the DE stage, and transcription activation occurred at early hepatic cell stage, were categorized as ‘late genes’ (Supplementary Figure S3A and C); genes whose expression levels were concomitantly up-regulated with Foxa2 binding at the DE stage, such as Foxa2, Gata4, and nine other genes, were grouped as ‘early genes’ (Supplementary Figure S3B and D). Histone H3 acetylation (H3ac) status on all of these genes in DE cells was first examined. H3ac around Foxa2 binding sites could only be detected on the ‘early genes’, but not on the ‘late genes’ (Supplementary Figure S4A). Next, histone H3 methylation status on these genes was examined. High levels of both H3K4me2 and H3K27me3 were detected 420 | Journal of Molecular Cell Biology (2012), 4, 420–422 doi:10.1093/jmcb/mjs037 Published online June 28, 2012

Genome-wide roles of Foxa2 in directing liver specification.

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