PBRM-1/PBAF-regulated genes in a multipotent progenitor in C. elegans

The Caenorhabditiselegans somatic gonadal precursors (SGPs) are multipotent progenitors that generate all somatic cells of the adult reproductive system. The two SGPs originate in the mesodermal layer and are born through a division that produces one SGP and one head mesodermal cell (hmc). One hmc terminally differentiates and the other dies by programmed cell death. The PBAF chromatin remodeling complex promotes the multipotent SGP fate. Complete loss of PBAF causes lethality, so we used a combination of Cre/lox recombination and GFP nanobody-directed protein degradation to eliminate PBRM-1, the signature subunit of the PBAF complex, from 83 mesodermal cells, including SGPs, body muscles, and the hmc. We used RNA sequencing to identify genes acting downstream of PBAF in these cells and identified 1955 transcripts that were significantly differentially expressed between pbrm-1(-) and pbrm-1(+) in the mesoderm of L1 larvae. Two strains were compared for this study. One strain uses the hnd-1 promoter to drive Cre/lox recombination and GFP nanobody-directed protein degradation in mesodermal tissues; this is pbrm-1(TS-KO). A control strain containing all the selectable markers found in pbrm-1(TS-KO) but lacking the GFP nanobody and Cre expression was used for comparisons, this is prbrm-1(control). L1 larvae were collected from pbrm-1(TS-KO) and pbrm-1(control). Five replicates were performed. Collections were made in parallel, on different days. RNA sequencing was used to identify differentially expressed genes between pbrm-1(TS-KO) and pbrm-1(control).

秀丽隐杆线虫（Caenorhabditis elegans）的体细胞生殖前体细胞（somatic gonadal precursors, SGPs）是一类多能祖细胞，可生成成体生殖系统的全部体细胞。两个SGPs起源于中胚层，通过一次分裂产生一个SGP与一个头部中胚层细胞（head mesodermal cell, hmc）；其中一个hmc会发生终末分化，另一个则通过程序性细胞死亡凋亡。PBAF染色质重塑复合物（PBAF chromatin remodeling complex）可促进SGP的多能性命运。由于完全缺失PBAF会导致个体致死，本研究结合Cre/lox重组技术与GFP纳米抗体介导的蛋白质降解技术，在包括SGPs、体肌及hmc在内的83个中胚层细胞中清除PBAF复合物的标志性亚基PBRM-1。本研究通过RNA测序（RNA sequencing）鉴定上述细胞中PBAF下游的作用基因，在L1期幼虫的中胚层内，共鉴定出1955个在pbrm-1(-)与pbrm-1(+)之间显著差异表达的转录本。本研究共比较两个品系：一个品系借助hnd-1启动子在中胚层组织中驱动Cre/lox重组与GFP纳米抗体介导的蛋白质降解，记为pbrm-1(TS-KO)；另一个为对照品系，其携带pbrm-1(TS-KO)中所有筛选标记，但缺失GFP纳米抗体与Cre的表达，记为pbrm-1(control)。研究人员收集了pbrm-1(TS-KO)与pbrm-1(control)的L1期幼虫，共设置5次生物学重复，所有样本均于不同日期平行收集。后续通过RNA测序鉴定pbrm-1(TS-KO)与pbrm-1(control)之间的差异表达基因。