Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA.
Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.
Genetics. 2022 Jul 4;221(3). doi: 10.1093/genetics/iyac073.
The mammalian pocket protein family, which includes the Retinoblastoma protein (pRb) and Rb-like pocket proteins p107 and p130, regulates entry into and exit from the cell cycle by repressing cell cycle gene expression. Although pRb plays a dominant role in mammalian systems, p107 and p130 are the ancestral pocket proteins. The Rb-like pocket proteins interact with the highly conserved 5-subunit MuvB complex and an E2F-DP transcription factor heterodimer, forming the DREAM (for Dp, Rb-like, E2F, and MuvB) complex. DREAM complex assembly on chromatin culminates in repression of target genes mediated by the MuvB subcomplex. Here, we examined how the Rb-like pocket protein contributes to DREAM formation and function by disrupting the interaction between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit LIN-52 using CRISPR/Cas9 targeted mutagenesis. A triple alanine substitution of LIN-52's LxCxE motif severed LIN-35-MuvB association and caused classical DREAM mutant phenotypes, including synthetic multiple vulvae, high-temperature arrest, and ectopic expression of germline genes in the soma. However, RNA-sequencing revealed limited upregulation of DREAM target genes when LIN-35-MuvB association was severed, as compared with gene upregulation following LIN-35 loss. Based on chromatin immunoprecipitation, disrupting LIN-35-MuvB association did not affect the chromatin localization of E2F-DP, LIN-35, or MuvB components. In a previous study, we showed that in worms lacking LIN-35, E2F-DP, and MuvB chromatin occupancy was reduced genome-wide. With LIN-35 present but unable to associate with MuvB, our study suggests that the E2F-DP-LIN-35 interaction promotes E2F-DP's chromatin localization, which we hypothesize supports MuvB chromatin occupancy indirectly through DNA. Altogether, this study highlights how the pocket protein's association with MuvB supports DREAM function but is not required for DREAM's chromatin occupancy.
哺乳动物口袋蛋白家族包括视网膜母细胞瘤蛋白（pRb）和 pRb 样口袋蛋白 p107 和 p130，通过抑制细胞周期基因的表达来调节细胞周期的进入和退出。虽然 pRb 在哺乳动物系统中发挥主导作用，但 p107 和 p130 是祖先口袋蛋白。Rb 样口袋蛋白与高度保守的 5 亚基 MuvB 复合物和 E2F-DP 转录因子异二聚体相互作用，形成 DREAM（Dp、Rb 样、E2F 和 MuvB）复合物。染色质上 DREAM 复合物的组装最终导致 MuvB 亚基复合物介导的靶基因的抑制。在这里，我们通过使用 CRISPR/Cas9 靶向诱变破坏了秀丽隐杆线虫唯一的口袋蛋白 LIN-35 与 MuvB 亚基 LIN-52 之间的相互作用，研究了 Rb 样口袋蛋白如何有助于 DREAM 的形成和功能。LIN-52 的 LxCxE 基序的三个丙氨酸取代物切断了 LIN-35-MuvB 之间的联系，并导致了经典的 DREAM 突变表型，包括多阴门、高温阻滞和生殖细胞基因在体中的异位表达。然而，与 LIN-35 缺失后基因的上调相比，当 LIN-35-MuvB 之间的联系被切断时，只有有限的 DREAM 靶基因被上调。基于染色质免疫沉淀，破坏 LIN-35-MuvB 之间的联系并不影响 E2F-DP、LIN-35 或 MuvB 成分的染色质定位。在之前的一项研究中，我们表明在缺乏 LIN-35 的蠕虫中，E2F-DP 和 MuvB 染色质占有率在全基因组范围内降低。当 LIN-35 存在但不能与 MuvB 结合时，我们的研究表明，E2F-DP-LIN-35 相互作用促进了 E2F-DP 的染色质定位，我们假设这通过 DNA 间接支持 MuvB 染色质占有率。总的来说，这项研究强调了口袋蛋白与 MuvB 的结合如何支持 DREAM 的功能，但不是 DREAM 的染色质占有率所必需的。
