IRX in epidermal differentiation

提案摘要 表皮分化对健康至关重要，儿童皮肤病的分化缺陷， 包括常见的特应性皮炎表皮分化是一个复杂的过程， 表观遗传变化和转录因子协调调节结构蛋白的表达， 粘附分子和表皮屏障形成所必需的脂质产生酶。戏剧基因 在分化中观察到的表达变化对应于表观遗传调节因子的大规模重组 结构，如超级增强子，优先驱动对细胞身份至关重要的基因的表达。在 最近的实验中，我们研究了增强子形成的动态过程中分化的人， 角质形成细胞在包含Grhl 3基因体的超级增强子内，Grhl 3是表皮细胞生长的关键调节因子。 分化，我们确定了丰富的结合位点的IRX，一个转录因子与未知的作用， 表皮，表明IRX可能促进表皮分化。与这个想法一致，我们发现， 小鼠表皮发育中几种IRX因子的时间表达反映了已知的 调节表皮分化的转录因子，包括GRHL 3。GRHL 3的缺失 角质形成细胞改变了增强子景观，形成了新的非角质形成细胞超级增强子， 促进神经元细胞的命运。有趣的是，这些新形成的增强子表现出惊人的过度表达， IRX结合位点，表明GRHL 3可能抑制角质形成细胞表现出神经元型胶原的倾向。 功能.小鼠表皮发育中几种Irx基因的时间表达模式与 即众所周知的表皮分化。新生小鼠表皮的单细胞分析揭示了梯度 Irx 3的表达与角质形成细胞的分化程度相关。这些初步结果 表明IRX在表皮分化中起关键作用。假设是， GRHL 3和IRX通过至少两种不同的机制促进表皮分化：1）IRX和GRHL 3均通过两种不同的机制促进表皮分化。 GRHL 3激活表达表皮分化因子的基因，和2）GRHL 3抑制表皮分化因子的形成。 IRX敏感的SE，否则将激活分化中的非表皮神经元基因， 表皮提出了两个目的来检验假设：目的1将利用人的体外模型， 表皮角质形成细胞分化提供了深入了解的机制， 分化目的2将利用条件性Irx 3; Irx 5双敲除小鼠来表征表皮 在表皮中缺乏Irx 3和Irx 5的小鼠的分化表型。这项工作将确立 IRX在皮肤发育中的作用，并阐明GRHL 3和IRX如何在表皮分化中协作。

PROPOSAL SUMMARY Epidermal differentiation is critical for health with differentiation defects underlying skin diseases in children, including the common Atopic Dermatitis. Epidermal differentiation is a complex process in which enhancers, epigenetic changes, and transcription factors coordinately regulate the expression of structural proteins, adhesion molecules, and lipid producing enzymes necessary for epidermal barrier formation. Dramatic gene expression changes observed in differentiation correspond to large-scale reorganization of epigenetic regulator structures such as super-enhancers, which preferentially drive expression of genes vital for cell identity. In recent experiments, we have studied the dynamics of enhancer formation during differentiation of human keratinocytes. Within the super-enhancer encompassing the gene body of Grhl3, a key regulator of epidermal differentiation, we identified enriched binding sites for IRX, a transcription factor with unknown roles in the epidermis, suggesting that IRX may promote epidermal differentiation. Consistent with this idea, we found that the temporal expression of several IRX factors in mouse epidermal development mirrors that of known regulatory transcription factors for epidermal differentiation, including GRHL3. Depletion of GRHL3 in keratinocytes changes the enhancer landscape with formation of new non-keratinocyte super-enhancers that promote neuronal cell fate. Intriguingly, these newly formed enhancers exhibit striking overrepresentation of IRX binding sites, suggesting that GRHL3 may suppress the tendency of keratinocytes to exhibit neuronal-type features. The temporal expression pattern of several Irx genes in mouse epidermal development is similar to that of well-known epidermal differentiation. Single cell analysis of newborn mice epidermis unveiled a gradient of Irx3 expression that correlates with the keratinocyte's degree of differentiation. These preliminary results suggest that IRX plays a key role in epidermal differentiation. The hypothesis is that an interplay between GRHL3 and IRX promotes epidermal differentiation through at least two different mechanisms: 1) both IRX and GRHL3 activate genes expressing epidermal differentiation factors, and 2) GRHL3 suppresses the formation of IRX-sensitive SEs that otherwise would activate non-epidermal, neuronal genes in the differentiating epidermis. Two Aims are proposed to test the hypothesis: Aim 1 will utilize an in vitro model of human epidermal keratinocyte differentiation to provide insights into the mechanism whereby IRX regulates differentiation. Aim 2 will utilize conditional Irx3;Irx5 double knockout mice to characterize the epidermal differentiation phenotype of mice deficient for Irx3 and Irx5 in the epidermis. This work will establish the role of IRX in skin development and elucidate how GRHL3 and IRX collaborate in epidermal differentiation.