Young LINE-1 transposon 5' UTRs marked by elongation factor ELL3 function as enhancers to regulate naïve pluripotency in embryonic stem cells.

LINE-1s are the major clade of retrotransposons with autonomous retrotransposition activity. Despite the potential genotoxicity, LINE-1s are highly activated in early embryos. Here we show that a subset of young LINE-1s, L1Md_Ts, are marked by the RNA polymerase II elongation factor ELL3, and function as enhancers in mouse embryonic stem cells. ELL3 depletion dislodges the DNA hydroxymethylase TET1 and the co-repressor SIN3A from L1Md_Ts, but increases the enrichment of the Bromodomain protein BRD4, leading to loss of 5hmC, gain of H3K27ac, and upregulation of the L1Md_T nearby genes. Specifically, ELL3 occupies and represses the L1Md_T-based enhancer located within Akt3, which encodes a key regulator of AKT pathway. ELL3 is required for proper ERK activation and efficient shutdown of naïve pluripotency through inhibiting Akt3 during naïve-primed transition. Our study reveals that the enhancer function of a subset of young LINE-1s controlled by ELL3 in transcription regulation and mouse early embryo development.

Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation.

The super elongation complex (SEC) containing P-TEFb plays a critical role in regulating transcription elongation. AFF1 and AFF4, members of the AF4/FMR2 family, act as central scaffold proteins of SEC and are associated with various human diseases. However, their precise roles in transcriptional control remain unclear. We here reveal differences in the genomic distribution patterns of AFF1 and AFF4 around transcription start sites (TSSs). AFF1 mainly binds upstream of the TSSs, while AFF4 is enriched downstream of the TSSs. Notably, disruption of AFF4 results in slow elongation and early termination in a subset of AFF4 bound active genes, whereas AFF1 deletion leads to fast elongation and transcriptional readthrough in the same gene subset. Additionally, AFF1 knockdown increases AFF4 levels at chromatin, and vice versa. In summary, these findings demonstrate that AFF1 and AFF4 function antagonistically to regulate Pol II transcription.

ZFP281-BRCA2 prevents R-loop accumulation during DNA replication.

R-loops are prevalent in mammalian genomes and involved in many fundamental cellular processes. Depletion of BRCA2 leads to aberrant R-loop accumulation, contributing to genome instability. Here, we show that ZFP281 cooperates with BRCA2 in preventing R-loop accumulation to facilitate DNA replication in embryonic stem cells. ZFP281 depletion reduces PCNA levels on chromatin and impairs DNA replication. Mechanistically, we demonstrate that ZFP281 can interact with BRCA2, and that BRCA2 is enriched at G/C-rich promoters and requires both ZFP281 and PRC2 for its proper recruitment to the bivalent chromatin at the genome-wide scale. Furthermore, depletion of ZFP281 or BRCA2 leads to accumulation of R-loops over the bivalent regions, and compromises activation of the developmental genes by retinoic acid during stem cell differentiation. In summary, our results reveal that ZFP281 recruits BRCA2 to the bivalent chromatin regions to ensure proper progression of DNA replication through preventing persistent R-loops.

RNF219 interacts with CCR4-NOT in regulating stem cell differentiation.

Regulation of RNA stability plays a crucial role in gene expression control. Deadenylation is the initial rate-limiting step for the majority of RNA decay events. Here, we show that RING finger protein 219 (RNF219) interacts with the CCR4-NOT deadenylase complex. RNF219-CCR4-NOT exhibits deadenylation activity in vitro. RNA-seq analyses identify some of the 2-cell-specific genes and the neuronal genes significantly downregulated upon RNF219 knockdown, while upregulated after depletion of the CCR4-NOT subunit CNOT10 in mouse embryonic stem (ES) cells. RNF219 depletion leads to impaired neuronal lineage commitment during ES cell differentiation. Our study suggests that RNF219 is a novel interacting partner of CCR4-NOT and required for maintenance of ES cell pluripotency.

ENL initiates multivalent phase separation of the super elongation complex (SEC) in controlling rapid transcriptional activation.

Release of paused RNA polymerase II (Pol II) requires incorporation of the positive transcription elongation factor b (P-TEFb) into the super elongation complex (SEC), thus resulting in rapid yet synchronous transcriptional activation. However, the mechanism underlying dynamic transition of P-TEFb from inactive to active state remains unclear. Here, we found that the SEC components are able to compartmentalize and concentrate P-TEFb via liquid-liquid phase separation from the soluble inactive HEXIM1 containing the P-TEFb complex. Specifically, ENL or its intrinsically disordered region is sufficient to initiate the liquid droplet formation of SEC. AFF4 functions together with ENL in fluidizing SEC droplets. SEC droplets are fast and dynamically formed upon serum exposure and required for rapid transcriptional induction. We also found that the fusion of ENL with MLL can boost SEC phase separation. In summary, our results suggest a critical role of multivalent phase separation of SEC in controlling transcriptional pause release.