研究基础：We constructed a 4-layer wood formation transcriptional regulatory network (TRN) in black cottonwood (Populus trichocarpa) that has four Secondary wall-associated NAC-Domain 1 (PtrSND1) transcription factor (TF) family members as the top-layer regulators.

主要研究：We characterized the function of a MYB (PtrMYB161) TF in this PtrSND1-TRN, using transgenic P. trichocarpa cells and whole plants.

结果1-过表达：PtrMYB161 is a third-layer regulator that directly transactivates five wood formation genes. Overexpression of PtrMYB161 in P. trichocarpa (OE-PtrMYB161s) led to reduced wood, altered cell type proportions, and inhibited growth.

结果2-负反馈：Integrative analysis of wood cell-based chromatin binding assays with OE-PtrMYB161s transcriptomics revealed a feedback regulation system in the PtrSND1-TRN, where PtrMYB161 represses all four top-layer regulators and one second-layer regulator, PtrMYB021, possibly affecting many downstream TFs in, and likely beyond, the TRN, to generate the observed phenotypic changes.

结果3-抑制功能：Our data also suggested that the PtrMYB161's repressor function operates through interaction of the base PtrMYB161-target binding system with gene silencing co-factors. PtrMYB161 protein does not contain any known negative regulatory domains.

结果4-突变：CRISPR-based mutants of PtrMYB161in P. trichocarpa exhibited phenotypes similar to the wildtype, suggesting PtrMYB161's activator functions are redundant among many TFs.

结论：Our work demonstrated that PtrMYB161 binds to multiple sets of target genes, a feature that allows it to function as an activator as well as a repressor. The balance of the two functions may be important to the establishment of regulatory homeostasis for normal growth and development.

木材形成是一个十分复杂的过程，涉及细胞分化、细胞扩张、次生细胞壁沉积以及程序化细胞死亡。作者在之前的研究中构建了一个四层木材形成转录调控网络（TRN），其中四个次生细胞壁相关的PtrSND1转录因子家族成员是顶层的调控因子。本文中，作者利用转基因毛果杨细胞和植株，研究了转录因子PtrMYB161在该PtrSND1-TRN转录调控网络中的作用。PtrMYB161是第三层的调控因子，直接反式激活5个木材形成相关的基因。在毛果杨中过表达PtrMYB161会导致木材减少、细胞类型的比例发生改变，并且会抑制植株生长。结合基于木材细胞的染色质结合试验以及OE-PtrMYB161s转录组分析，作者发现了一个PtrSND1-TRN的反馈调节系统，其中PtrMYB161会抑制所有四个顶层调控因子以及一个第二层调控因子PtrMYB021，同时也有可能影响着许多下游的转录因子，从而影响表型的变化。本文的研究还显示，PtrMYB161的抑制功能是通过基础的PtrMYB161-靶标结合系统与基因沉默辅因子的相互作用而起作用的。PtrMYB161蛋白并不包含任何已知的负调控结构域。基于CRISPR的毛果杨PtrMYB161突变体的表型特征与野生型类似，说明PtrMYB161的激活功能与其它许多转录因子功能冗余。本文的研究揭示了PtrMYB161结合多个靶基因，使得其即能作为激活子，也可以作为抑制子。而PtrMYB161激活与抑制功能之间的平衡对于正常的生长与发育调控内稳态的建立是非常重要的。