FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

好文献非常难得，提供了核心的研究思路。

FTO Obesity Variant Circuitry and Adipocyte Browning in Humans - 这篇文章需要好好的解析

为深入研究一个non-coding variant的作用机制提供了完整的思路

本文宏伟的目标：we sought to identify a causal variant with regulatory roles, its upstream regulator, and its downstream target gene in order to provide a candidate mechanistic basis for the association between FTO and obesity.

回头点评：

• 这篇文章，方法无所不用其极，最终搞出了一个完整的故事
• GWAS的数据是核心的立足点，挖掘里面的common的casual variant
• non coding variant的研究思路
• 自下而上的研究思路，先确定核心的基因，在把基因和casual variant串起来

基本概念

FTO是一个基因，与肥胖有显著关联，GWAS分析也鉴定出了一些显著的variant。

Fat mass and obesity-associated protein also known as alpha-ketoglutarate-dependent dioxygenase FTO is an enzyme that in humans is encoded by the FTO gene located on chromosome 16.

肥胖与其他疾病的关系，contributes to type 2 diabetes, cardiovascular disorders, and cancer

身体质量指数（BMI，Body Mass Index）是国际上常用的衡量人体肥胖程度和是否健康的重要标准，主要用于统计分析。

Haplotype-specific enhancer assays，可以检测enhancer在指定haplotype里面的活性

long-range three-dimensional chromatin interactions，研究enhancer的target genes的必备武器

cis-eQTL是指近距离相关的eQTL, trans-eQTL则是包括了远距离相关的eQTL。这两类eQTL对应 cis-regulation以及 trans-regulation。由于trans-eQTL的计算涉及到更大量的多重检验，因此我们采取了更严格的数据质量控制。

phylogenetic module complexity analysis (PMCA)，找casual variant

结果

EFFECT OF THE FTO LOCUS ON IRX3 AND IRX5 IN HUMAN ADIPOCYTE PROGENITOR CELLS

首先需要确定causal variant在哪个组织中发挥作用

然后就要预测这个enhancer的target gene

方法：找genotype-associated expression的基因，就是这个enhancer里面的risk allele与基因关联在一起了，有可能就是target gene（这是研究non-coding variant和target gene的有效手段）

确定了组织：the effect was cell type–specific and restricted to preadipocytes, which represent a minority of cells in adipose tissue

确定了两个candidate gene：IRX3 and IRX5

EFFECT OF THE FTO LOCUS ON MITOCHONDRIAL THERMOGENESIS AND LIPID STORAGE

确定IRX3 and IRX5涉及的biologic processes

方法：在健康人中确定positively or negatively correlated with IRX3 and IRX5 expression的基因

问题：这里只有10个人，核心要义是co-expression，问题是会找到很多关联的基因，这里明显就是经过筛选的，不管怎样，这样的结果只可以的。

因为全部是负相关，所以推测是repressor。these findings indicated potential roles for IRX3 and IRX5 in the repression of thermogenesis

examine the trans-eQTL genetic control of energy balance by the FTO obesity locus，远距离的eQTL

As compared with nonrisk-allele carriers, risk-allele carriers had lower expression of mitochondrial, browning, and respiration genes and higher expression of lipid-storage markers, which indicated a shift from energy dissipation to energy storage.

ADIPOCYTE-AUTONOMOUS EFFECTS OF IRX3 AND IRX5 ON ENERGY BALANCE

直接用基因敲出来直接推测基因的作用

IRX3 and IRX5 levels recapitulate the effect that the FTO genetic variant has on thermogenesis

上小鼠实验，Irx3 dominant-negative (aP2-Irx3DN) mice

组织自治和大脑介入，tissue-autonomous versus brain-mediated roles

Irx3 and Irx5 have cell-autonomous roles

DETERMINATION OF THE CAUSAL VARIANT AND DISRUPTION OF REPRESSION BY ARID5B

开始找causal variant了，自下而上，非常好！！！

To predict the causal variant, the disruption of which is necessary and sufficient to cause IRX3 and IRX5 dysregulation in human preadipocytes

phylogenetic module complexity analysis (PMCA)找到了rs1421085这个casual variant

a gain of enhancer activity in association with the rs1421085 risk allele

To evaluate the effect of the variant on regulator binding, we used electrophoretic mobility-shift assays (EMSAs)

通过motif的变化，找到了上游的调控基因

We examined disrupted motifs and regulator expression to identify potential upstream regulators

evaluate the causal role of ARID5B

结论，故事圆满

the FTO obesity variant acts through disruption of ARID5B binding in the risk haplotype, leading to a loss of repression, a gain of enhancer activity, and increases in IRX3 and IRX5 expression

C-TO-T EDITING OF THE RS1421085 RISK VARIANT AND THE EFFECT ON THERMOGENESIS

Targeted genome editing technology involving CRISPR–Cas9登场

最终验证

文章逻辑非常严密，研究思路非常科学，值得借鉴，N Engl J Med也不是那么的遥不可及！！！

待看：

Leveraging Cross-Species Transcription Factor Binding Site Patterns: From Diabetes Risk Loci to Disease Mechanisms

有需要以后还会再看

待续~