Orlovsky Kira, Appel Elena, Hantisteanu Shay, Olender Tsviya, Lotem Joseph, Levanon Ditsa, Groner Yoram
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
PLoS Genet. 2024 Dec 23;20(12):e1011401. doi: 10.1371/journal.pgen.1011401. eCollection 2024 Dec.
The development and diversification of sensory proprioceptive neurons, which reside in the dorsal root ganglia (DRG) and express the tropomyosin receptor kinase C (TrkC), depend on the transcription factor (TF) Runx3. Runx3-deficient mice develop severe limb ataxia due to TrkC neuron cell death. Two additional TFs Pou4f1 (also called Brn3a) and Isl1 also play an important role in sensory neuron development. Thus, we aimed to unravel the chromatin state of early-developing TrkC neurons and decipher the Runx3 high-confidence target genes (HCT) and the possible cooperation between Runx3, Brn3a and Isl1 in the regulation of these genes.
Runx3 expression is driven by the gene proximal P2 promoter. Transcriptome analysis was conducted by RNA-seq on RNA isolated from heterozygous (P2+/-) vs. homozygous (P2-/-) TrkC neurons and differentially expressed genes (DEGs) were determined. Genome-wide occupancy of Runx3, Brn3a, Isl1 and histone H3 acetylated on lysine 27 (H3K27Ac) was determined using CUT&RUN. The landscape of Transposase-accessible chromatin was analyzed via ATAC-seq.
The intersection of Runx3 genomic occupancy-associated genes and DEG data discovered 244 Runx3 HCT. Brn3a and Isl1 were found to bind to numerous genomic loci, some of which overlapped with Runx3. Most genomic regions bound by each of these three TFs or co-bound by them resided in distantly located enhancer regions rather than in gene promoters. In activated and suppressed neuronal Runx3 HCT, Runx3 cooperated mainly with Brn3a to regulate expression through distantly located enhancers. Interestingly, suppression of non-neuronal immune genes was mainly managed via Runx3 without Brn3a. The distribution of ATAC and H3K27Ac marked regions in Runx3 peaks containing at least one RUNX binding site (Runx3_RBS) revealed that while most promoter regions were marked by ATAC, a prominent fraction of intron/intergenic regions occupied by Runx3, Brn3a or Isl1 were unmarked by ATAC and/or H3K27Ac.
These analyses shed new light on the interplay of Runx3, Brn3a, Isl1, and open chromatin regions in regulating the Runx3 HCT in the early developmental stages of TrkC neurons.
感觉本体感觉神经元位于背根神经节（DRG）并表达原肌球蛋白受体激酶C（TrkC），其发育和多样化依赖于转录因子（TF）Runx3。Runx3基因缺陷型小鼠由于TrkC神经元细胞死亡而出现严重的肢体共济失调。另外两个转录因子Pou4f1（也称为Brn3a）和Isl1在感觉神经元发育中也发挥重要作用。因此，我们旨在揭示早期发育的TrkC神经元的染色质状态，破译Runx3高可信度靶基因（HCT）以及Runx3、Brn3a和Isl1在这些基因调控中的可能协作关系。
Runx3的表达由基因近端P2启动子驱动。对从杂合子（P2+/-）与纯合子（P2-/-）TrkC神经元中分离的RNA进行RNA测序以进行转录组分析，并确定差异表达基因（DEG）。使用CUT&RUN确定Runx3、Brn3a、Isl1和赖氨酸27乙酰化组蛋白H3（H3K27Ac）的全基因组占有率。通过ATAC测序分析转座酶可及染色质景观。
Runx3基因组占有率相关基因与DEG数据的交集发现了244个Runx3 HCT。发现Brn3a和Isl1与众多基因组位点结合，其中一些与Runx3重叠。这三种转录因子各自结合或共同结合的大多数基因组区域位于远距离的增强子区域而非基因启动子中。在激活和抑制的神经元Runx3 HCT中，Runx3主要与Brn3a协作通过远距离增强子调节表达。有趣的是，非神经元免疫基因的抑制主要通过Runx3而无需Brn3a来调控。在含有至少一个RUNX结合位点（Runx3_RBS）的Runx3峰中，ATAC和H3K27Ac标记区域的分布表明，虽然大多数启动子区域被ATAC标记，但Runx3、Brn3a或Isl1占据的内含子/基因间区域中有相当一部分未被ATAC和/或H3K27Ac标记。
这些分析为Runx3、Brn3a、Isl1和开放染色质区域在TrkC神经元早期发育阶段调控Runx3 HCT中的相互作用提供了新的见解。
